Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

PubMed

Dybwad, Marius; van der Laaken, Anton L; Blatny, Janet Martha; Paauw, Armand

2013-09-01

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

PubMed Central

van der Laaken, Anton L.; Blatny, Janet Martha; Paauw, Armand

2013-01-01

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 106 spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min. PMID:23811517

Survival of Bacillus anthracis spores in fruit juices and wine.

PubMed

Leishman, Oriana N; Johnson, Miranda J; Labuza, Theodore P; Diez-Gonzalez, Francisco

2010-09-01

Foods have been identified as a potential target for bioterrorism due to their essential nature and global distribution. Foods produced in bulk have the potential to have large batches of product intentionally contaminated, which could affect hundreds or thousands of individuals. Bacillus anthracis spores are one potential bioterrorism agent that may survive pasteurization and remain viable throughout the shelf life of fruit juices and cause disease if consumed. This project examined B. anthracis spore survival in orange, apple, and grape juices, as well as wine. Samples of beverages were inoculated with spores of two nonpathogenic B. anthracis strains at approximately 10(6) CFU/ml, and the spore count was determined periodically during storage for 30 days at 4°C. After this time, the counts of survival spores never declined more than 1 log CFU/ml in any of the beverage types. These results indicate that spores can survive, with little to no loss in viability, for at least a month in fruit juices and wine.

Anthrax Detection: Agencies Need to Validate Sampling Activities in Order to Increase Confidence in Negative Results

DTIC Science & Technology

2005-03-01

validate all activities related to other biothreat agents. In September and October 2001, letters laced with Bacillus anthracis (anthrax) spores were...2001, contaminated letters laced with Bacillus anthracis, or anthrax spores ,1 were sent through the mail to two senators, Thomas Daschle and Patrick...equipped workforce collecting the environmental samples; maximized isolation of viable Bacillus anthracis through preservation of spores during transport

The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid.

PubMed

Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

2016-01-01

Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 10(4) spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites.

The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid

PubMed Central

Celebi, Ozgur; Buyuk, Fatih; Pottage, Tom; Crook, Ant; Hawkey, Suzanna; Cooper, Callum; Bennett, Allan; Sahin, Mitat; Baillie, Leslie

2016-01-01

Elimination of Bacillus anthracis spores from the environment is a difficult and costly process due in part to the toxicity of current sporicidal agents. For this reason we investigated the ability of the spore germinants L-alanine (100 mM) and inosine (5 mM) to reduce the concentration of peracetic acid (PAA) required to inactivate B. anthracis spores. While L-alanine significantly enhanced (p = 0.0085) the bactericidal activity of 500 ppm PAA the same was not true for inosine suggesting some form of negative interaction. In contrast the germinant combination proved most effective at 100 ppm PAA (p = 0.0009). To determine if we could achieve similar results in soil we treated soil collected from the burial site of an anthrax infected animal which had been supplemented with spores of the Sterne strain of B. anthracis to increase the level of contamination to 104 spores/g. Treatment with germinants followed 1 h later by 5000 ppm PAA eliminated all of the spores. In contrast direct treatment of the animal burial site using this approach delivered using a back pack sprayer had no detectable effect on the level of B. anthracis contamination or on total culturable bacterial numbers over the course of the experiment. It did trigger a significant, but temporary, reduction (p < 0.0001) in the total spore count suggesting that germination had been triggered under real world conditions. In conclusion, we have shown that the application of germinants increase the sensitivity of bacterial spores to PAA. While the results of the single field trial were inconclusive, the study highlighted the potential of this approach and the challenges faced when attempting to perform real world studies on B. anthracis spores contaminated sites. PMID:26858699

Inactivation of Bacillus anthracis Spores during Laboratory-Scale Composting of Feedlot Cattle Manure

PubMed Central

Xu, Shanwei; Harvey, Amanda; Barbieri, Ruth; Reuter, Tim; Stanford, Kim; Amoako, Kingsley K.; Selinger, Leonard B.; McAllister, Tim A.

2016-01-01

Anthrax outbreaks in livestock have social, economic and health implications, altering farmer’s livelihoods, impacting trade and posing a zoonotic risk. Our study investigated the survival of Bacillus thuringiensis and B. anthracis spores sporulated at 15, 20, or 37°C, over 33 days of composting. Spores (∼7.5 log10 CFU g-1) were mixed with manure and composted in laboratory scale composters. After 15 days, the compost was mixed and returned to the composter for a second cycle. Temperatures peaked at 71°C on day 2 and remained ≥55°C for an average of 7 days in the first cycle, but did not exceed 55°C in the second. For B. thuringiensis, spores generated at 15 and 21°C exhibited reduced (P < 0.05) viability of 2.7 and 2.6 log10 CFU g-1 respectively, as compared to a 0.6 log10 CFU g-1 reduction for those generated at 37°C. For B. anthracis, sporulation temperature did not impact spore survival as there was a 2.5, 2.2, and 2.8 log10 CFU g-1 reduction after composting for spores generated at 15, 21, and 37°C, respectively. For both species, spore viability declined more rapidly (P < 0.05) in the first as compared to the second composting cycle. Our findings suggest that the duration of thermophilic exposure (≥55°C) is the main factor influencing survival of B. anthracis spores in compost. As sporulation temperature did not influence survival of B. anthracis, composting may lower the viability of spores associated with carcasses infected with B. anthracis over a range of sporulation temperatures. PMID:27303388

Decontamination of fluid milk containing Bacillus spores using commercial household products.

PubMed

Black, D G; Taylor, T M; Kerr, H J; Padhi, S; Montville, T J; Davidson, P M

2008-03-01

Although commercial sanitizers can inactivate bacterial spores in food processing environments, relatively little data exist as to the decontamination of products and surfaces by consumers using commercial household products. Should a large scale bioterrorism incident occur in which consumer food products were contaminated with a pathogenic sporeformer such as Bacillus anthracis, there may be a need to decontaminate these products before disposal as liquid or solid waste. Studies were conducted to test the efficacy of commercial household products for inactivating spores of Bacillus cereus (used as a surrogate for B. anthracis) in vitro and in fluid milk. Validation of the resistance of the B. cereus spores was confirmed with B. anthracis spores. Fifteen commercial products, designed as either disinfectants or sanitizers or as potential sanitizers, were purchased from retail markets. Products selected had one of the following active compounds: NaOCl, HCl, H2O2, acetic acid, quaternary ammonium compounds, ammonium hydroxide, citric acid, isopropanol, NaOH, or pine oil. Compounds were diluted in water (in vitro) or in 2% fat fluid milk, and spores were exposed for up to 6 h. Products containing hypochlorite were most effective against B. cereus spores. Products containing HCl or H2O2 also reduced significant numbers of spores but at a slower rate. The resistance of spores of surrogate B. cereus strains to chlorine-containing compounds was similar to that of B. anthracis spores. Therefore, several household products on the market may be used to decontaminate fluid milk or similar food products contaminated by spores of B. anthracis.

A Simple Decontamination Approach Using Hydrogen ...

EPA Pesticide Factsheets

Journal article To evaluate the use of relatively low levels of hydrogen peroxide vapor (HPV) for the inactivation of Bacillus anthracis spores within an indoor environment. Methods and Results: Laboratory-scale decontamination tests were conducted using bacterial spores of both B. anthracis Ames and Bacillus atrophaeus inoculated onto several types of materials. Pilot-scale tests were also conducted using a larger chamber furnished as an indoor office. Commercial off-the-shelf (COTS) humidifiers filled with aqueous solutions of 3% or 8% hydrogen peroxide were used to generate the HPV inside the mock office. The spores were exposed to the HPV for periods ranging from 8 hours up to one week. Conclusions: Four to seven day exposures to low levels of HPV (average air concentrations of approximately 5-10 parts per million) were effective in inactivating B. anthracis spores on multiple materials. The HPV can be generated with COTS humidifiers and household H2O2 solutions. With the exception of one test/material, B. atrophaeus spores were equally or more resistant to HPV inactivation compared to those from B. anthracis Ames. Significance and Impact of Study: This simple and effective decontamination method is another option that could be widely applied in the event of a B. anthracis spore release.

FORMALDEHYDE GAS INACTIVATION OF BACILLUS ANTHRACIS, BACILLUS SUBTILIS AND GEOBACILLUS STEAROTHERMOPHILUS SPORES ON INDOOR SURFACE MATERIALS.

EPA Science Inventory

Research evaluated the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface material using formaldehyde gas. Spores were dried on seven types of indoor surfaces and exposed to 1100 ppm formaldehyde gas for 10 hr. Fo...

The effect of growth medium on B. anthracis Sterne spore carbohydrate content.

PubMed

Colburn, Heather A; Wunschel, David S; Antolick, Kathryn C; Melville, Angela M; Valentine, Nancy B

2011-06-01

The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation. Copyright © 2011 Elsevier B.V. All rights reserved.

Inactivation of Bacillus anthracis Spores in Soil Matrices with ...

EPA Pesticide Factsheets

Report This report documents the results of a laboratory study designed to better understand the effectiveness of chlorine dioxide (ClO2) gas to decontaminate soil materials contaminated with Bacillus anthracis spores.

Most Probable Number Rapid Viability PCR Method to Detect Viable Spores of Bacillus anthracis in Swab Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Letant, S E; Kane, S R; Murphy, G A

2008-05-30

This note presents a comparison of Most-Probable-Number Rapid Viability (MPN-RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs generated by the Centers for Disease Control and Prevention (CDC) for a multi-center validation study aimed at testing environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were in statistical agreement with the CDC conventional culture method for all three levels of spores tested (10{sup 4}, 10{sup 2}, and 10 spores) even in the presence ofmore » dirt. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols.« less

Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples

PubMed Central

Ramage, Jason G.; Prentice, Kristin W.; DePalma, Lindsay; Venkateswaran, Kodumudi S.; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R.; Sharma, Shashi; Estacio, Peter L.; Stanker, Larry; Hodge, David R.

2016-01-01

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert® test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 106 spores/mL (ca. 1.5 × 105 spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores. PMID:27661796

Trace detection of meglumine and diatrizoate from Bacillus spore samples using liquid chromatography/mass spectrometry.

PubMed

Swider, Catherine; Maguire, Kelly; Rickenbach, Michael; Montgomery, Madeline; Ducote, Matthew J; Marhefka, Craig A

2012-07-01

Following the September 11, 2001 terrorist attacks, letters containing Bacillus anthracis were distributed through the United States postal system killing five people. A complex forensic investigation commenced to identify the perpetrator of these mailings. A novel liquid chromatography/mass spectrometry protocol for the qualitative detection of trace levels of meglumine and diatrizoate in dried spore preparations of B. anthracis was developed. Meglumine and diatrizoate are components of radiographic imaging products that have been used to purify bacterial spores. Two separate chromatographic assays using multiple mass spectrometric analyses were developed for the detection of meglumine and diatrizoate. The assays achieved limits of detection for meglumine and diatrizoate of 1.00 and 10.0 ng/mL, respectively. Bacillus cereus T strain spores were effectively used as a surrogate for B. anthracis spores during method development and validation. This protocol was successfully applied to limited evidentiary B. anthracis spore material, providing probative information to the investigators. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples.

PubMed

Thomas, M C; Shields, M J; Hahn, K R; Janzen, T W; Goji, N; Amoako, K K

2013-07-01

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10(1) and 1·3 × 10(2)  CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples. © Her Majesty the Queen in Right of Canada [2013]. Reproduced with the permission of the Canadian Food Inspection Agency.

Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope.

PubMed

Hutchison, Janine R; Erikson, Rebecca L; Sheen, Allison M; Ozanich, Richard M; Kelly, Ryan T

2015-09-21

Bacillus anthracis is the causative agent of anthrax and can be contracted by humans and herbivorous mammals by inhalation, ingestion, or cutaneous exposure to bacterial spores. Due to its stability and disease potential, B. anthracis is a recognized biothreat agent and robust detection and viability methods are needed to identify spores from unknown samples. Here we report the use of smartphone-based microscopy (SPM) in combination with a simple microfluidic incubation device (MID) to detect 50 to 5000 B. anthracis Sterne spores in 3 to 5 hours. This technique relies on optical monitoring of the conversion of the ∼1 μm spores to the filamentous vegetative cells that range from tens to hundreds of micrometers in length. This distinguishing filament formation is unique to B. anthracis as compared to other members of the Bacillus cereus group. A unique feature of this approach is that the sample integrity is maintained, and the vegetative biomass can be removed from the chip for secondary molecular analysis such as PCR. Compared with existing chip-based and rapid viability PCR methods, this new approach reduces assay time by almost half, and is highly sensitive, specific, and cost effective.

Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis

PubMed Central

Stratilo, Chad W.; Crichton, Melissa K. F.; Sawyer, Thomas W.

2015-01-01

Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes. PMID:26394165

Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

PubMed

Plaut, Roger D; Staab, Andrea B; Munson, Mark A; Gebhardt, Joan S; Klimko, Christopher P; Quirk, Avery V; Cote, Christopher K; Buhr, Tony L; Rossmaier, Rebecca D; Bernhards, Robert C; Love, Courtney E; Berk, Kimberly L; Abshire, Teresa G; Rozak, David A; Beck, Linda C; Stibitz, Scott; Goodwin, Bruce G; Smith, Michael A; Sozhamannan, Shanmuga

2018-04-01

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure".

PubMed

Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Fleming, Joy; Bi, Li-Jun; Yang, Rui-Fu; Zhang, Xian-En

2015-05-15

Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Identifying experimental surrogates for Bacillus anthracis spores: a review

PubMed Central

2010-01-01

Bacillus anthracis, the causative agent of anthrax, is a proven biological weapon. In order to study this threat, a number of experimental surrogates have been used over the past 70 years. However, not all surrogates are appropriate for B. anthracis, especially when investigating transport, fate and survival. Although B. atrophaeus has been widely used as a B. anthracis surrogate, the two species do not always behave identically in transport and survival models. Therefore, we devised a scheme to identify a more appropriate surrogate for B. anthracis. Our selection criteria included risk of use (pathogenicity), phylogenetic relationship, morphology and comparative survivability when challenged with biocides. Although our knowledge of certain parameters remains incomplete, especially with regards to comparisons of spore longevity under natural conditions, we found that B. thuringiensis provided the best overall fit as a non-pathogenic surrogate for B. anthracis. Thus, we suggest focusing on this surrogate in future experiments of spore fate and transport modelling. PMID:21092338

Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores

PubMed Central

Wendling, Morgan; Richter, William; Lastivka, Andrew; Mickelsen, Leroy

2016-01-01

The primary goal of this study was to determine the conditions required for the effective inactivation of Bacillus anthracis spores on materials by using methyl bromide (MeBr) gas. Another objective was to obtain comparative decontamination efficacy data with three avirulent microorganisms to assess their potential for use as surrogates for B. anthracis Ames. Decontamination tests were conducted with spores of B. anthracis Ames and Geobacillus stearothermophilus, B. anthracis NNR1Δ1, and B. anthracis Sterne inoculated onto six different materials. Experimental variables included temperature, relative humidity (RH), MeBr concentration, and contact time. MeBr was found to be an effective decontaminant under a number of conditions. This study highlights the important role that RH has when fumigation is performed with MeBr. There were no tests in which a ≥6-log10 reduction (LR) of B. anthracis Ames was achieved on all materials when fumigation was done at 45% RH. At 75% RH, an increase in the temperature, the MeBr concentration, or contact time generally improved the efficacy of fumigation with MeBr. This study provides new information for the effective use of MeBr at temperatures and RH levels lower than those that have been recommended previously. The study also provides data to assist with the selection of an avirulent surrogate for B. anthracis Ames spores when additional tests with MeBr are conducted. PMID:26801580

Humanized theta-defensins (retrocyclins) enhance macrophage performance and protect mice from experimental anthrax infections.

PubMed

Welkos, S; Cote, C K; Hahn, U; Shastak, O; Jedermann, J; Bozue, J; Jung, G; Ruchala, P; Pratikhya, P; Tang, T; Lehrer, R I; Beyer, W

2011-09-01

Retrocyclins are humanized versions of the -defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by "piggyback phagocytosis," model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.

Measurement of 100 B. anthracis Ames spores within 15 minutes by SERS at the US Army Edgewood Chemical Biological Ctr.

NASA Astrophysics Data System (ADS)

Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

2014-05-01

Since the distribution of Bacillus anthracis-Ames spores through the US Postal System, there has been a persistent fear that biological warfare agents will be used by terrorists against our military abroad and our civilians at home. While there has been substantial effort since the anthrax attack of 2001 to develop analyzers to detect this and other biological warfare agents, the analyzers remain either too slow, lack sensitivity, produce high false-positive rates, or cannot be fielded. In an effort to overcome these limitations we have been developing a surface-enhanced Raman spectroscopy system. Here we describe the use of silver nanoparticles functionalized with a short peptide to selectively capture Bacillus anthracis spores and produce SER scattering. Specifically, measurements of 100 B. anthracis-Ames spores/mL in ~25 minutes performed at the US Army's Edgewood Chemical Biological Center are presented. The measurements provide a basis for the development of systems that can detect spores collected from the air or water supplies with the potential of saving lives during a biological warfare attack.

Requirements for the Development of Bacillus Anthracis Spore Reference Materials Used to Test Detection Systems

PubMed Central

Almeida, Jamie L.; Wang, Lili; Morrow, Jayne B.; Cole, Kenneth D.

2006-01-01

Bacillus anthracis spores have been used as biological weapons and the possibility of their further use requires surveillance systems that can accurately and reliably detect their presence in the environment. These systems must collect samples from a variety of matrices, process the samples, and detect the spores. The processing of the sample may include removal of inhibitors, concentration of the target, and extraction of the target in a form suitable for detection. Suitable reference materials will allow the testing of each of these steps to determine the sensitivity and specificity of the detection systems. The development of uniform and well-characterized reference materials will allow the comparison of different devices and technologies as well as assure the continued performance of detection systems. This paper discusses the special requirements of reference materials for Bacillus anthracis spores that could be used for testing detection systems. The detection of Bacillus anthracis spores is based on recognition of specific characteristics (markers) on either the spore surface or in the nucleic acids (DNA). We have reviewed the specific markers and their relevance to characterization of reference materials. We have also included the approach for the characterization of candidate reference materials that we are developing at the NIST laboratories. Additional applications of spore reference materials would include testing sporicidal treatments, techniques for sampling the environment, and remediation of spore-contaminated environments. PMID:27274929

[Survival of Bacillus anthracis spores in various tannery baths].

PubMed

Mendrycka, M; Mierzejewski, J

2000-01-01

The influence of tannery baths: liming, deliming, bating, pickling, tanning, retannage on the survival and on the germination dynamism of B. anthracis spores (Sterne strain) was investigated. The periods and the conditions of this influence were established according to technological process of cow hide tannage. Practically after every bath some part of the spores remained vital. The most effective killing of spores occurred after pickling, liming and deliming. Inversely, the most viable spores remained after bating and retannage process. The lack of correlation that was observed between survival and germination of spores after retannage bath can be explained by different mechanism of spores germination inhibition and their killing.

Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection

PubMed Central

Cote, Christopher K.; Van Rooijen, Nico; Welkos, Susan L.

2006-01-01

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. PMID:16369003

Aerosol and Surface Deposition Characteristics of Two Surrogates for Bacillus anthracis Spores

PubMed Central

Stapleton, Helen L.

2016-01-01

ABSTRACT Spores of an acrystalliferous derivative of Bacillus thuringiensis subsp. kurstaki, termed Btcry−, are morphologically, aerodynamically, and structurally indistinguishable from Bacillus anthracis spores. Btcry− spores were dispersed in a large, open-ended barn together with spores of Bacillus atrophaeus subsp. globigii, a historically used surrogate for Bacillus anthracis. Spore suspensions (2 × 1012 CFU each of B. atrophaeus subsp. globigii and Btcry−) were aerosolized in each of five spray events using a backpack misting device incorporating an air blower; a wind of 4.9 to 7.6 m s−1 was also flowing through the barn in the same direction. Filter air samplers were situated throughout the barn to assess the aerosol density of the spores during each release. Trays filled with a surfactant in aqueous buffer were placed on the floor near the filter samplers to assess spore deposition. Spores were also recovered from arrays of solid surfaces (concrete, aluminum, and plywood) that had been laid on the floor and set up as a wall at the end of the barn. B. atrophaeus subsp. globigii spores were found to remain airborne for significantly longer periods, and to be deposited on horizontal surfaces at lower densities, than Btcry− spores, particularly near the spray source. There was a 6-fold-higher deposition of Btcry− spores than of B. atrophaeus subsp. globigii spores on vertical surfaces relative to the surrounding airborne density. This work is relevant for selecting the best B. anthracis surrogate for the prediction of human exposure, hazard assessment, and hazard management following a malicious release of B. anthracis. IMPORTANCE There is concern that pathogenic bacteria could be maliciously disseminated in the air to cause human infection and disruption of normal life. The threat from spore-forming organisms, such as the causative agent of anthrax, is particularly serious. In order to assess the extent of this risk, it is important to have a surrogate organism that can be used to replicate the dispersal characteristics of the threat agent accurately. This work compares the aerosol dispersal and deposition behaviors of the surrogates Btcry− and B. atrophaeus subsp. globigii. Btcry− spores remained in the air for a shorter time, and were markedly more likely to adhere to vertical surfaces, than B. atrophaeus subsp. globigii spores. PMID:27613681

Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid.

PubMed

Buhr, T L; Wells, C M; Young, A A; Minter, Z A; Johnson, C A; Payne, A N; McPherson, D C

2013-08-01

To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants. Spores (≥ 7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2.7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid. Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials. A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

ENVIRONMENTAL RESPONSE TO INTENTIONAL DISSEMINATION OF BACILLUS ANTHRACIS SPORES IN THE UNITED STATES--2001

EPA Science Inventory

The intentional dissemination of Bacillus anthracis (anthrax) spores at multiple locations in the United States in the Fall of 2001 resulted not only in several deaths and illnesses (including psychological effects), but likely changed lifestyles and attitudes, and increased the ...

Bacillus anthracis Interacts with Plasmin(ogen) to Evade C3b-Dependent Innate Immunity

PubMed Central

Chung, Myung-Chul; Tonry, Jessica H.; Narayanan, Aarthi; Manes, Nathan P.; Mackie, Ryan S.; Gutting, Bradford; Mukherjee, Dhritiman V.; Popova, Taissia G.; Kashanchi, Fatah; Bailey, Charles L.; Popov, Serguei G.

2011-01-01

The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen. PMID:21464960

Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores

PubMed Central

Edmonds, Jason; Lindquist, H. D. Alan; Sabol, Jonathan; Martinez, Kenneth; Shadomy, Sean; Cymet, Tyler; Emanuel, Peter

2016-01-01

The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening. PMID:27123934

Disinfection of Vegetative Cells of Bacillus anthracis

DTIC Science & Technology

2016-03-01

1. INTRODUCTION Disinfection of Bacillus anthracis spores in drinking water is well documented in peer-reviewed literature (Adcock et al., 2004... Disinfection kinetics of vegetative cells of Bacillus anthracis in water with free available chlorine ([FAC] 2 mg/L) and monochloramine ([MC] 2 mg/L) were...anthracis. Bacillus anthracis cells Drinking water Chlorine demand-free (CDF

Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

PubMed

Delvecchio, Vito G; Connolly, Joseph P; Alefantis, Timothy G; Walz, Alexander; Quan, Marian A; Patra, Guy; Ashton, John M; Whittington, Jessica T; Chafin, Ryan D; Liang, Xudong; Grewal, Paul; Khan, Akbar S; Mujer, Cesar V

2006-09-01

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.

Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis‡

PubMed Central

DelVecchio, Vito G.; Connolly, Joseph P.; Alefantis, Timothy G.; Walz, Alexander; Quan, Marian A.; Patra, Guy; Ashton, John M.; Whittington, Jessica T.; Chafin, Ryan D.; Liang, Xudong; Grewal, Paul; Khan, Akbar S.; Mujer, Cesar V.

2006-01-01

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development. PMID:16957262

Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

PubMed

Buhr, T L; Young, A A; Minter, Z A; Wells, C M; McPherson, D C; Hooban, C L; Johnson, C A; Prokop, E J; Crigler, J R

2012-11-01

To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. Hot, humid air is a potential alternative to conventional chemical decontamination. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

PubMed Central

Nerandzic, Michelle M.; Rackaityte, Elze; Jury, Lucy A.; Eckart, Kevin; Donskey, Curtis J.

2013-01-01

Background Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe®) would reduce the burden of spores on skin. Methods Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths. Results Spore removal from hands was enhanced with Vashe soak (>2.5 log10 reduction) versus soap and water wash or soak (~2.0 log10 reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log10 spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin. Conclusions Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin. PMID:23844234

Pilot-scale crossflow-microfiltration and pasturization to remove spores of Bacillus anthracis (Sterne) from milk

USDA-ARS?s Scientific Manuscript database

HTST pasteurization of milk is generally ineffective against spore-forming bacteria such as Bacillus anthracis (BA) but is lethal to its vegetative cells. Crossflow microfiltration (MF), using ceramic membranes with a pore diameter of 1.4 um, has been shown to physically remove somatic cells, vegeta...

Glycoconjugates as Mediators of Nitric Oxide Production upon Exposure to Bacterial Spores by Macrophages

NASA Astrophysics Data System (ADS)

Lahiani, Mohamed; Soderberg, Lee; Tarasenko, Olga

2011-06-01

Phagocytes generate nitric oxide (NO) in large quantities to combat bacteria. The spore-producing Gram-positive organisms of Bacillus cereus family are causative agents from mild to a life threatening infection in humans and domestic animals. Our group have shown that glycoconjugates (GCs) activate macrophages and enhance killing of Bacillus spores. In this investigation, we will explore the effect of different GCs structures on NO production. The objective of this study is to study effects of GCs 2, 4, 6, 8, 10 on NO release upon exposure to B. cereus and Bacillus anthracis spores by macrophages. Our results demonstrated that GCs activated macrophages and increased NO production using studied GCs ligands compared to macrophage only (p<0.001). GC2 and GC8 were able to further increase NO production in macrophages compared to the B. anthracis spores treated macrophages (p<0.001). Our finding suggests that GCs could be used as potential mediators of NO production in macrophages to fight B. anthracis and other pathogens.

Killed but metabolically active Bacillus anthracis vaccines induce broad and protective immunity against anthrax.

PubMed

Skoble, Justin; Beaber, John W; Gao, Yi; Lovchik, Julie A; Sower, Laurie E; Liu, Weiqun; Luckett, William; Peterson, Johnny W; Calendar, Richard; Portnoy, Daniel A; Lyons, C Rick; Dubensky, Thomas W

2009-04-01

Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

Sporulation and germination gene expression analysis of Bacillus anthracis Sterne spores in skim milk under heat and different intervention techniques

USDA-ARS?s Scientific Manuscript database

To investigate how B. anthracis Stene spores survive in milk under heat (80 degree C, 10 minutes), pasteurization (72 degree C, 15 seconds) and pasteurization plus microfiltration, the expression levels of genes that related to sporulation and germination were tested using real-time PCR assays. Tw...

Multigeneration Cross Contamination of Mail with Bacillus Species Spores by Tumbling ▿

PubMed Central

Edmonds, Jason; Clark, Paul; Williams, Leslie; Lindquist, H. D. Alan; Martinez, Kenneth; Gardner, Warren; Shadomy, Sean; Hornsby-Myers, Jennifer

2010-01-01

In 2001, envelopes loaded with Bacillus anthracis spores were mailed to Senators Daschle and Leahy as well as to the New York Post and NBC News buildings. Additional letters may have been mailed to other news agencies because there was confirmed anthrax infection of employees at these locations. These events heightened the awareness of the lack of understanding of the mechanism(s) by which objects contaminated with a biological agent might spread disease. This understanding is crucial for the estimation of the potential for exposure to ensure the appropriate response in the event of future attacks. In this study, equipment to simulate interactions between envelopes and procedures to analyze the spread of spores from a “payload” envelope (i.e., loaded internally with a powdered spore preparation) onto neighboring envelopes were developed. Another process to determine whether an aerosol could be generated by opening contaminated envelopes was developed. Subsequent generations of contaminated envelopes originating from a single payload envelope showed a consistent two-log decrease in the number of spores transferred from one generation to the next. Opening a tertiary contaminated envelope resulted in an aerosol containing 103 B. anthracis spores. We developed a procedure for sampling contaminated letters by a nondestructive method aimed at providing information useful for consequence management while preserving the integrity of objects contaminated during the incident and preserving evidence for law enforcement agencies. PMID:20511424

High-Throughput Detection of Bacillus Anthracis Spores using Peptide-Conjugated Nano/Micro-Beads

DTIC Science & Technology

2006-07-26

natto was a lab-stock isolated from natto . Expertise with B. anthracis ∆Sterne (pXO1-, pXO2-) and B. anthracis Sterne 34F2 (pXO1+, pXO2-) were...spore-peptide-Qdot complexes were analyzed by FACS. We found that BA1 peptide did not bind to B. subtilis DB104, B. subtilis natto and B. cereus...subtilis DB104, B. subtilis natto and B. cereus used as other more negative controls did not show fluorescence (data not shown). We then examined the

Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

DTIC Science & Technology

2015-06-19

animal waste an~ decompositiOn DISTRIBUTION STATEMENT A: Approved for public release; distribution is unlimited. UNCLASSIFIED PR-15-306 Anthrax...influx of water. Ungerminated spore Germination Germinated spore Spore hydratation ~ Non-refractile spore Refractile spore • Fluorescence

Disinfection methods for spores of Bacillus atrophaeus, B. anthracis, Clostridium tetani, C. botulinum and C. difficile.

PubMed

Oie, Shigeharu; Obayashi, Akiko; Yamasaki, Hirofumi; Furukawa, Hiroyuki; Kenri, Tsuyoshi; Takahashi, Motohide; Kawamoto, Keiko; Makino, Sou-ichi

2011-01-01

To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.

Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, Janine R.; Erikson, Rebecca L.; Sheen, Allison M.

Rapid, cost-effective bacterial detection systems are needed to respond to potential biothreat events. Here we report the use of smartphone-based microscopy in combination with a simple microfluidic incubation device to detect 5000 Bacillus anthracis spores in 3 hours. This field-deployable approach is compatible with real-time PCR for secondary confirmation.

Optimization of a sample processing protocol for recovery of Bacillus anthracis spores from soil

USGS Publications Warehouse

Silvestri, Erin E.; Feldhake, David; Griffin, Dale; Lisle, John T.; Nichols, Tonya L.; Shah, Sanjiv; Pemberton, A; Schaefer III, Frank W

2016-01-01

Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps. Optimization of the protocol included: identifying an ideal extraction diluent, variation in the number of wash steps, variation in the initial centrifugation speed, sonication and shaking mechanisms. The optimized protocol was demonstrated at two laboratories in order to evaluate the recovery of spores from loamy and sandy soils. The new protocol demonstrated an improved limit of detection for loamy and sandy soils over the non-optimized protocol with an approximate matrix limit of detection at 14 spores/g of soil. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol will be robust enough to use at multiple laboratories while achieving comparable recoveries.

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

DOE PAGES

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.; ...

2015-07-30

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

PubMed Central

Wood, Joseph P.; Meyer, Kathryn M.; Kelly, Thomas J.; Choi, Young W.; Rogers, James V.; Riggs, Karen B.; Willenberg, Zachary J.

2015-01-01

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log10 reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially. PMID:26372011

Scanning Surface Potential Microscopy of Spore Adhesion on Surfaces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ida; Chung, Eunhyea; Kweon, Hyojin

2012-01-01

The adhesion of spores of Bacillus anthracis - the cause of anthrax and a likely biological threat - to solid surfaces is an important consideration in cleanup after an accidental or deliberate release. However, because of safety concerns, directly studying B. anthracis spores with advanced instrumentation is problematic. As a first step, we are examining the electrostatic potential of Bacillus thuringiensis (Bt), which is a closely related species that is often used as a simulant to study B. anthracis. Scanning surface potential microscopy (SSPM), also known as Kelvin probe force microscopy (KPFM), was used to investigate the influence of relativemore » humidity (RH) on the surface electrostatic potential of Bt that had adhered to silica, mica, or gold substrates. AFM/SSPM side-by-side images were obtained separately in air, at various values of RH, after an aqueous droplet with spores was applied on each surface and allowed to dry before measurements. In the SSPM images, a negative potential on the surface of the spores was observed compared with that of the substrates. The surface potential decreased as the humidity increased. Spores were unable to adhere to a surface with an extremely negative potential, such as mica.« less

Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown

PubMed Central

Goossens, Pierre L.; Tournier, Jean-Nicolas

2015-01-01

Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signaling, thereby paralyzing the immune response of the host and perturbing the endocrine and endothelial systems. In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia. PMID:26500645

Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores

PubMed Central

Bernhards, Casey B.

2014-01-01

The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup. PMID:25022853

Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry

PubMed Central

Waller, David F.; Hew, Brian E.; Holdaway, Charlie; Jen, Michael; Peckham, Gabriel D.

2016-01-01

Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field. PMID:27999382

Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture.

PubMed

Hutchison, J R; Piepel, G F; Amidan, B G; Hess, B M; Sydor, M A; Deatherage Kaiser, B L

2018-05-01

We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials and assay methods on false-negative rate (FNR) and limit of detection (LOD 95 ) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2-500 per coupon) onto glass, stainless steel, vinyl tile and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD 95 results. Mean FNRs tended to be lower for mRV-PCR compared to culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD 95 was lowest for glass and highest for vinyl tile. LOD 95 values overall were lower for mRV-PCR than for the culture method. This study adds to the limited data on FNR and LOD 95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis. © 2018 The Society for Applied Microbiology.

The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants

PubMed Central

March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A

2015-01-01

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking. PMID:26185111

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Bok, Jasper M; van Rotterdam, Bart J

2010-12-08

Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

PubMed Central

2010-01-01

Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

Human anthrax as a re-emerging disease.

PubMed

Doganay, Mehmet; Demiraslan, Hayati

2015-01-01

Anthrax is primarily a disease of herbivores and the etiological agent is B. anthracis which is a gram-positive, aerobic, spore-forming, and rod shaped bacterium. Bacillus anthracis spores are highly resistant to heat, pressure, ultraviolet and ionizing radiation, chemical agents and disinfectants. For these reasons, B. anthracis spores are an attractive choice as biological agents for the use of bioweapon and/or bioterrorism. Soil is the main reservoir for the infectious agent. The disease most commonly affects wild and domestic mammals. Human are secondarily infected by contact with infected animals and contaminated animal products or directly expose to B. anthracis spores. Anthrax occurs worldwide. This infection is still endemic or hyperendemic in both animals and humans in some part of areas of the world; particularly in Middle East, West Africa, Central Asia, some part of India, South America. However, some countries are claiming free of anthrax, and anthrax has become a re-emerging disease in western countries with the intentional outbreak. Currently, anthrax is classified according to its setting as (1) naturally occurring anthrax, (2) bioterrorism-related anthrax. Vast majority of human anthrax are occurring as naturally occurring anthrax in the world. It is also a threaten disease for western countries. The aim of this paper is to review the relevant patents, short historical perspective, microbiological and epidemiological features, clinical presentations and treatment.

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

PubMed

Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

2015-02-01

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies ▿

PubMed Central

Raber, Ellen; Burklund, Alison

2010-01-01

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (102 and 106 spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 102 spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult. PMID:20709855

Anthrax: A disease of biowarfare and public health importance

PubMed Central

Goel, Ajay Kumar

2015-01-01

Bioterrorism has received a lot of attention in the first decade of this century. Biological agents are considered attractive weapons for bioterrorism as these are easy to obtain, comparatively inexpensive to produce and exhibit widespread fear and panic than the actual potential of physical damage. Bacillus anthracis (B. anthracis), the etiologic agent of anthrax is a Gram positive, spore forming, non-motile bacterium. This is supposed to be one of the most potent BW agents because its spores are extremely resistant to natural conditions and can survive for several decades in the environment. B. anthracis spores enter the body through skin lesion (cutaneous anthrax), lungs (pulmonary anthrax), or gastrointestinal route (gastrointestinal anthrax) and germinate, giving rise to the vegetative form. Anthrax is a concern of public health also in many countries where agriculture is the main source of income including India. Anthrax has been associated with human history for a very long time and regained its popularity after Sept 2001 incidence in United States. The present review article describes the history, biology, life cycle, pathogenicity, virulence, epidemiology and potential of B. anthracis as biological weapon. PMID:25610847

Test methods and response surface models for hot, humid air decontamination of materials contaminated with dirty spores of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam.

PubMed

Buhr, T L; Young, A A; Barnette, H K; Minter, Z A; Kennihan, N L; Johnson, C A; Bohmke, M D; DePaola, M; Cora-Laó, M; Page, M A

2015-11-01

To develop test methods and evaluate survival of Bacillus anthracis ∆Sterne or Bacillus thuringiensis Al Hakam on materials contaminated with dirty spore preparations after exposure to hot, humid air using response surface modelling. Spores (>7 log10 ) were mixed with humic acid + spent sporulation medium (organic debris) or kaolin (dirt debris). Spore samples were then dried on five different test materials (wiring insulation, aircraft performance coating, anti-skid, polypropylene, and nylon). Inoculated materials were tested with 19 test combinations of temperature (55, 65, 75°C), relative humidity (70, 80, 90%) and time (1, 2, 3 days). The slowest spore inactivation kinetics was on nylon webbing and/or after addition of organic debris. Hot, humid air effectively decontaminates materials contaminated with dirty Bacillus spore preparations; debris and material interactions create complex decontamination kinetic patterns; and B. thuringiensis Al Hakam is a realistic surrogate for B. anthracis. Response surface models of hot, humid air decontamination were developed which may be used to select decontamination parameters for contamination scenarios including aircraft. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions.

PubMed

Koehler, Susanne M; Buyuk, Fatih; Celebi, Ozgur; Demiraslan, Hayati; Doganay, Mehmet; Sahin, Mitat; Moehring, Jens; Ndumnego, Okechukwu C; Otlu, Salih; van Heerden, Henriette; Beyer, Wolfgang

2017-07-12

Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores

PubMed Central

Perry, K. Allison; O’Connell, Heather A.; Rose, Laura J.; Noble-Wang, Judith A.; Arduino, Matthew J.

2016-01-01

The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at −15°C, 5°C, 21°C, or 35°C for 0–7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T0) was determined for each variable. No differences were observed in recovery between swabs held at −15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 102, p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at −15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores. PMID:27213119

Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores.

PubMed

Perry, K Allison; O'Connell, Heather A; Rose, Laura J; Noble-Wang, Judith A; Arduino, Matthew J

The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis . Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T 0 ) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10 2 , p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores

PubMed Central

2014-01-01

Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256

Protocol for Detection of Bacillus anthracis in Environmental Samples

EPA Pesticide Factsheets

This pProtocol Method describes proceduresintended for the analyses of swabs, wipes, Sponge-Sticks, vacuum socks and filters, air filters, drinking water, and decontamination waste water for Bacillus anthracis spores.

Thermal Inactivation of Bacillus anthracis Spores Using Rapid Resistive Heating

DTIC Science & Technology

2016-03-24

thermal inactivation research. However, the research conducted to support this thesis utilizes the B.a. Sterne strain which is used in livestock vaccines...methodology conducted for this research including hard surface recovery, thermal inactivation of Bacillus anthracis spores, and the rapid resistive heating...to 500°C range but again, many of the thermal inactivation studies were conducted in the 350 to 2000°C range. Sample plots will be discussed in

Species specific identification of spore-producing microbes using the gene sequence of small acid-soluble spore coat proteins for amplification based diagnostics

DOEpatents

McKinney, Nancy

2002-01-01

PCR (polymerase chain reaction) primers for the detection of certain Bacillus species, such as Bacillus anthracis. The primers specifically amplify only DNA found in the target species and can distinguish closely related species. Species-specific PCR primers for Bacillus anthracis, Bacillus globigii and Clostridium perfringens are disclosed. The primers are directed to unique sequences within sasp (small acid soluble protein) genes.

Immunization studies with attenuated strains of Bacillus anthracis.

PubMed Central

Ivins, B E; Ezzell, J W; Jemski, J; Hedlund, K W; Ristroph, J D; Leppla, S H

1986-01-01

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. PMID:3084383

Comparison of false-negative rates and limits of detection following macrofoam-swab sampling of Bacillus anthracis surrogates via Rapid Viability PCR and plate culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, J. R.; Piepel, G. F.; Amidan, B. G.

Aims: We evaluated the effects of Bacillus anthracis surrogates, low surface concentrations, surface materials, and assay methods on false-negative rate (FNR) and limit of detection (LOD95) for recovering Bacillus spores using a macrofoam-swab sampling procedure. Methods and Results: Bacillus anthracis Sterne or Bacillus atrophaeus Nakamura spores were deposited over a range of low target concentrations (2 – 500 coupon-1) onto glass, stainless steel, vinyl tile, and plastic. Samples were assayed using a modified Rapid Viability-PCR (mRV-PCR) method and the traditional plate culture method to obtain FNR and LOD95 results. Conclusions: Mean FNRs tended to be lower for mRV-PCR compared tomore » culturing, and increased as spore concentration decreased for all surface materials. Surface material, but not B. anthracis surrogate, influenced FNRs with the mRV-PCR method. The mRV-PCR LOD95 was lowest for glass and highest for vinyl tile. LOD95 values overall were lower for mRV-PCR than for the culture method. Significance and Impact of Study: This study adds to the limited data on FNR and LOD95 for mRV-PCR and culturing methods with low concentrations of B. anthracis sampled from various surface materials by the CDC macrofoam-swab method. These are key inputs for planning characterization and clearance studies for low contamination levels of B. anthracis.« less

In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

PubMed Central

Lambert, Emily A.; Sherry, Nora

2012-01-01

The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-δ-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of N-acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-δ-lactam. When the SleL derivatives are expressed in vivo, those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in B. anthracis sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified. PMID:22343356

Recovery of Bacillus Spore Contaminants from Rough Surfaces: a Challenge to Space Mission Cleanliness Control▿

PubMed Central

Probst, Alexander; Facius, Rainer; Wirth, Reinhard; Wolf, Marco; Moissl-Eichinger, Christine

2011-01-01

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load (“bioburden”) of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research. PMID:21216908

Achieving Consistent Multiple Daily Low-Dose Bacillus anthracis Spore Inhalation Exposures in the Rabbit Model

DTIC Science & Technology

2012-06-13

plethysmography. Overall, the presented results show that the animal aerosol system was stable and highly reproducible between different studies and over...develop and deliver low-doses of B. anthracis spores via inhalation in a reproducible manner. The pilot feasibility study (see Table 1 for results) enabled...results presented in Figure 3 show that exposures produced by the aerosol system were stable and reproducible from day-to-day. In all testing, the

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin

PubMed Central

Bartholomew, Rachel A.; Ozanich, Richard M.; Arce, Jennifer S.; Engelmann, Heather E.; Heredia-Langner, Alejandro; Hofstad, Beth A.; Hutchison, Janine R.; Jarman, Kristin; Melville, Angela M.; Victry, Kristin D.

2017-01-01

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 108 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 107 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use. PMID:28192054

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin.

PubMed

Bartholomew, Rachel A; Ozanich, Richard M; Arce, Jennifer S; Engelmann, Heather E; Heredia-Langner, Alejandro; Hofstad, Beth A; Hutchison, Janine R; Jarman, Kristin; Melville, Angela M; Victry, Kristin D; Bruckner-Lea, Cynthia J

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 10 8 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 10 7 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use.

The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

PubMed

March, Jordon K; Pratt, Michael D; Lowe, Chinn-Woan; Cohen, Marissa N; Satterfield, Benjamin A; Schaalje, Bruce; O'Neill, Kim L; Robison, Richard A

2015-10-01

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

PubMed Central

Bernhards, Casey B.; Chen, Yan; Toutkoushian, Hannah

2014-01-01

Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn2+ or Ca2+ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation. PMID:25384476

Anthrax Spores under a microscope

NASA Technical Reports Server (NTRS)

2003-01-01

Anthrax spores are inactive forms of Bacillus anthracis. They can survive for decades inside a spore's tough protective coating; they become active when inhaled by humans. A result of NASA- and industry-sponsored research to develop small greenhouses for space research is the unique AiroCide TiO2 system that kills anthrax spores and other pathogens.

Processing protocol for soil samples potentially contaminated with Bacillus anthracis spores [HS7.52.02 - 514

USGS Publications Warehouse

Silvestri, Erin E.; Griffin, Dale W.

2017-01-01

This protocol describes the processing steps for 45 g and 9 g soil samples potentially contaminated with Bacillus anthracis spores. The protocol is designed to separate and concentrate the spores from bulk soil down to a pellet that can be used for further analysis. Soil extraction solution and mechanical shaking are used to disrupt soil particle aggregates and to aid in the separation of spores from soil particles. Soil samples are washed twice with soil extraction solution to maximize recovery. Differential centrifugation is used to separate spores from the majority of the soil material. The 45 g protocol has been demonstrated by two laboratories using both loamy and sandy soil types. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol would be robust enough to use at multiple laboratories while achieving comparable recoveries. The 45 g protocol has demonstrated a matrix limit of detection at 14 spores/gram of soil for loamy and sandy soils.

Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

NASA Astrophysics Data System (ADS)

Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

2010-04-01

Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

PubMed Central

Ogawa, Hirohito; Fujikura, Daisuke; Ohnuma, Miyuki; Ohnishi, Naomi; Hang'ombe, Bernard M.; Mimuro, Hitomi; Ezaki, Takayuki; Mweene, Aaron S.; Higashi, Hideaki

2015-01-01

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis. PMID:25774512

Role of Visible Light-Activated Photocatalyst on the Reduction of Anthrax Spore-Induced Mortality in Mice

PubMed Central

Huang, Hsin-Hsien; Wong, Ming-Show; Lin, Hung-Chi; Chang, Hsin-Hou

2009-01-01

Background Photocatalysis of titanium dioxide (TiO2) substrates is primarily induced by ultraviolet light irradiation. Anion-doped TiO2 substrates were shown to exhibit photocatalytic activities under visible-light illumination, relative environmentally-friendly materials. Their anti-spore activity against Bacillus anthracis, however, remains to be investigated. We evaluated these visible-light activated photocatalysts on the reduction of anthrax spore-induced pathogenesis. Methodology/Principal Findings Standard plating method was used to determine the inactivation of anthrax spore by visible light-induced photocatalysis. Mouse models were further employed to investigate the suppressive effects of the photocatalysis on anthrax toxin- and spore-mediated mortality. We found that anti-spore activities of visible light illuminated nitrogen- or carbon-doped titania thin films significantly reduced viability of anthrax spores. Even though the spore-killing efficiency is only approximately 25%, our data indicate that spores from photocatalyzed groups but not untreated groups have a less survival rate after macrophage clearance. In addition, the photocatalysis could directly inactivate lethal toxin, the major virulence factor of B. anthracis. In agreement with these results, we found that the photocatalyzed spores have tenfold less potency to induce mortality in mice. These data suggest that the photocatalysis might injury the spores through inactivating spore components. Conclusion/Significance Photocatalysis induced injuries of the spores might be more important than direct killing of spores to reduce pathogenicity in the host. PMID:19132100

Biotechnology

NASA Image and Video Library

2003-01-22

Anthrax spores are inactive forms of Bacillus anthracis. They can survive for decades inside a spore's tough protective coating; they become active when inhaled by humans. A result of NASA- and industry-sponsored research to develop small greenhouses for space research is the unique AiroCide TiO2 system that kills anthrax spores and other pathogens.

False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, Janine R.; Piepel, Gregory F.; Amidan, Brett G.

Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, andmore » plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm2). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD95 was lowest for glass (0.429 CFU/cm2 with BAS and 0.341 CFU/cm2 with BG) and highest for vinyl tile (0.919 CFU/cm2 with BAS and 0.917 CFU/cm2 with BG). These mRV-PCR LOD95 values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm2 and BG: 0.820 to 1.489 CFU/cm2). The FNR and LOD95 values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.« less

False Negative Rates of a Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates via Real-Time PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, Janine R.; Piepel, Gregory F.; Amidan, Brett G.

Surface sampling for Bacillus anthracis spores has traditionally relied on detection via bacterial cultivation methods. Although effective, this approach does not provide the level of organism specificity that can be gained through molecular techniques. False negative rates (FNR) and limits of detection (LOD) were determined for two B. anthracis surrogates with modified rapid viability-polymerase chain reaction (mRV-PCR) following macrofoam-swab sampling. This study was conducted in parallel with a previously reported study that analyzed spores using a plate-culture method. B. anthracis Sterne (BAS) or B. atrophaeus Nakamura (BG) spores were deposited onto four surface materials (glass, stainless steel, vinyl tile, andmore » plastic) at nine target concentrations (2 to 500 spores/coupon; 0.078 to 19.375 colony-forming units [CFU] per cm²). Mean FNR values for mRV-PCR analysis ranged from 0 to 0.917 for BAS and 0 to 0.875 for BG and increased as spore concentration decreased (over the concentrations investigated) for each surface material. FNRs based on mRV-PCR data were not statistically different for BAS and BG, but were significantly lower for glass than for vinyl tile. FNRs also tended to be lower for the mRV-PCR method compared to the culture method. The mRV-PCR LOD₉₅ was lowest for glass (0.429 CFU/cm² with BAS and 0.341 CFU/cm² with BG) and highest for vinyl tile (0.919 CFU/cm² with BAS and 0.917 CFU/cm² with BG). These mRV-PCR LOD₉₅ values were lower than the culture values (BAS: 0.678 to 1.023 CFU/cm² and BG: 0.820 to 1.489 CFU/cm²). The FNR and LOD₉₅ values reported in this work provide guidance for environmental sampling of Bacillus spores at low concentrations.« less

Inactivation of Bacillus anthracis Spores by a Combination of Biocides and Heating under High-Temperature Short-Time Pasteurization Conditions ▿

PubMed Central

Xu, Sa; Labuza, Theodore P.; Diez-Gonzalez, Francisco

2008-01-01

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85°C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80°C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80°C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80°C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures. PMID:18390680

Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions.

PubMed

Xu, Sa; Labuza, Theodore P; Diez-Gonzalez, Francisco

2008-06-01

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.

Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis.

PubMed

Stokes, Margaret G M; Titball, Richard W; Neeson, Brendan N; Galen, James E; Walker, Nicola J; Stagg, Anthony J; Jenner, Dominic C; Thwaite, Joanne E; Nataro, James P; Baillie, Leslie W J; Atkins, Helen S

2007-04-01

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 10(5) CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.

Inadvertent laboratory exposure to Bacillus anthracis--California, 2004.

PubMed

2005-04-01

On June 9, 2004, the California Department of Health Services (CDHS) was notified of possible inadvertent exposure to Bacillus anthracis spores at Children's Hospital Oakland Research Institute (CHORI), where workers were evaluating the immune response of mice to B. anthracis. This report summarizes the subsequent investigation by CDHS and CDC, including assessment of exposures, administration of postexposure chemoprophylaxis, and serologic testing of potentially exposed workers. The findings underscore the importance of using appropriate biosafety practices and performing adequate sterility testing when working with material believed to contain inactivated B. anthracis organisms.

Evaluation of Surface Sampling for Bacillus Spores Using ...

EPA Pesticide Factsheets

Report The primary objectives of this project were to evaluate the Aggressive Air Sampling (AAS) method compared to currently used surface sampling methods and to determine if AAS is a viable option for sampling Bacillus anthracis spores.

Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

PubMed

Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

2017-10-03

Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

Requirements for the Development of Bacillus Anthracis Spore Reference Materials Used to Test Detection Systems

DTIC Science & Technology

2006-01-01

the sporangium) contributes the com- plex layers of the spore coats that encase the spore DNA. The mother cell dies and begins to fall apart at the end...spores. Bacillus spores contain a number of coat layers and some species posses an additional outermost layer called the exosporium. BA, B. cereus, and B...exosporium is the outermost layer of the BA spores, it likely contains important protein and carbohydrate markers that are recognized by antibodies

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

PubMed

Jain, Neha; Merwyn, S; Rai, G P; Agarwal, G S

2012-05-01

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in "real time" during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10(7) spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10(3) spores and 10(2) spores in talcum powder, respectively, whereas PCR could detect 10(4) spores in soil and 10(3) spores in talcum powder, respectively.

Evaluation of PCR Systems for Field Screening of Bacillus anthracis

PubMed Central

Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.; Bartholomew, Rachel A.; Arce, Jennifer S.; Heredia-Langner, Alejandro; Jarman, Kristin; Kreuzer, Helen W.

2017-01-01

There is little published data on the performance of hand-portable polymerase chain reaction (PCR) systems that can be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated 5 commercially available hand-portable PCR instruments for detection of Bacillus anthracis. We used a cost-effective, statistically based test plan to evaluate systems at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) of the probability of detection (POD) at confidence levels of 80% to 95%. We assessed specificity using purified genomic DNA from 13 B. anthracis strains and 18 Bacillus near neighbors, potential interference with 22 suspicious powders that are commonly encountered in the field by first responders during suspected biothreat incidents, and the potential for PCR inhibition when B. anthracis spores were spiked into these powders. Our results indicate that 3 of the 5 systems achieved 0.95 LCB of the probability of detection with 95% confidence levels at test concentrations of 2,000 genome equivalents/mL (GE/mL), which is comparable to 2,000 spores/mL. This is more than sufficient sensitivity for screening visible suspicious powders. These systems exhibited no false-positive results or PCR inhibition with common suspicious powders and reliably detected B. anthracis spores spiked into these powders, though some issues with assay controls were observed. Our testing approach enables efficient performance testing using a statistically rigorous and cost-effective test plan to generate performance data that allow users to make informed decisions regarding the purchase and use of field biodetection equipment. PMID:28192050

Next-Generation Bacillus anthracis Live Attenuated Spore Vaccine Based on the htrA- (High Temperature Requirement A) Sterne Strain

PubMed Central

Chitlaru, Theodor; Israeli, Ma’ayan; Bar-Haim, Erez; Elia, Uri; Rotem, Shahar; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

2016-01-01

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10–104-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization. PMID:26732659

Rapid Determination of Endospore Viability by Hyperspectral Reflectance Following Surface Decontamination

DTIC Science & Technology

2008-12-01

Alexandria, VA ABSTRACT Bacterial spores , or endospores, such as those of Bacillus anthracis, are an asymmetrical threat. Decontamination... Bacillus subtilis spores by hypochlorite and chlorine dioxide, J. Appl Microbiol., 95(1), 54-67. ...have the ability to distinguish viable from non-viable endospores. In the laboratory, we have exploited the oxidative alteration of the spore coat

Development of size-selective sampling of Bacillus anthracis surrogate spores from simulated building air intake mixtures for analysis via laser-induced breakdown spectroscopy.

PubMed

Gibb-Snyder, Emily; Gullett, Brian; Ryan, Shawn; Oudejans, Lukas; Touati, Abderrahmane

2006-08-01

Size-selective sampling of Bacillus anthracis surrogate spores from realistic, common aerosol mixtures was developed for analysis by laser-induced breakdown spectroscopy (LIBS). A two-stage impactor was found to be the preferential sampling technique for LIBS analysis because it was able to concentrate the spores in the mixtures while decreasing the collection of potentially interfering aerosols. Three common spore/aerosol scenarios were evaluated, diesel truck exhaust (to simulate a truck running outside of a building air intake), urban outdoor aerosol (to simulate common building air), and finally a protein aerosol (to simulate either an agent mixture (ricin/anthrax) or a contaminated anthrax sample). Two statistical methods, linear correlation and principal component analysis, were assessed for differentiation of surrogate spore spectra from other common aerosols. Criteria for determining percentages of false positives and false negatives via correlation analysis were evaluated. A single laser shot analysis of approximately 4 percent of the spores in a mixture of 0.75 m(3) urban outdoor air doped with approximately 1.1 x 10(5) spores resulted in a 0.04 proportion of false negatives. For that same sample volume of urban air without spores, the proportion of false positives was 0.08.

Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes

DTIC Science & Technology

2014-10-30

S1793984412300129 Marquita Lilly, Liju Yang, Kamal Aferchich. Effect of Single-walled Carbon Nanotubes on Bacillus Anthracis Cell Growth, Sporulation ...addition,  SWNTs  treatment  did  not  induce  sporulation  of B. anthracis.  [Aferichich, et al. 2012]. 2)  SWNTs  in  combination with oxidizing agents...8. Kamal Aferchich, Marquita Lilly, Liju Yang*. 2012. Effect of Single‐walled Carbon Nanotubes on  Bacillus Anthracis Cell Growth,  Sporulation , and

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model.

PubMed

Ndumnego, Okechukwu C; Köhler, Susanne M; Crafford, Jannie; van Heerden, Henriette; Beyer, Wolfgang

2016-10-01

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

EPA Science Inventory

Electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates were measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here prov...

Evaluation of peracetic acid fog for the inactivation of Bacillus anthracis spore surrogates in a large decontamination chamber.

PubMed

Wood, Joseph P; Calfee, Michael Worth; Clayton, Matthew; Griffin-Gatchalian, Nicole; Touati, Abderrahmane; Egler, Kim

2013-04-15

The purpose of this study was to evaluate the sporicidal (inactivation of bacterial spores) effectiveness and operation of a fogging device utilizing peracetic acid/hydrogen peroxide (PAA). Experiments were conducted in a pilot-scale 24 m(3) stainless steel chamber using either biological indicators (BIs) or bacterial spores deposited onto surfaces via aerosolization. Wipe sampling was used to recover aerosol-deposited spores from chamber surfaces and coupon materials before and after fogging to assess decontamination efficacy. Temperature, relative humidity, and hydrogen peroxide vapor levels were measured during testing to characterize the fog environment. The fog completely inactivated all BIs in a test using a 60 mL solution of PAA (22% hydrogen peroxide/4.5% peracetic acid). In tests using aerosol-deposited bacterial spores, the majority of the post-fogging spore levels per sample were less than 1 log colony forming units, with a number of samples having no detectable spores. In terms of decontamination efficacy, a 4.78 log reduction of viable spores was achieved on wood and stainless steel. Fogging of PAA solutions shows potential as a relatively easy to use decontamination technology in the event of contamination with Bacillus anthracis or other spore-forming infectious disease agents, although additional research is needed to enhance sporicidal efficacy. Published by Elsevier B.V.

Optimization of a Sample Processing Protocol for Recovery of ...

EPA Pesticide Factsheets

Journal Article Following a release of Bacillus anthracis spores into the environment, there is a potential for lasting environmental contamination in soils. There is a need for detection protocols for B. anthracis in environmental matrices. However, identification of B. anthracis within a soil is a difficult task. Processing soil samples helps to remove debris, chemical components, and biological impurities that can interfere with microbiological detection. This study aimed to optimize a previously used indirect processing protocol, which included a series of washing and centrifugation steps.

Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.

2015-08-07

Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extractionmore » improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.« less

Impacts of sporulation temperature, exposure to compost matrix and temperature on survival of Bacillus cereus spores during livestock mortality composting.

PubMed

Stanford, K; Reuter, T; Gilroyed, B H; McAllister, T A

2015-04-01

To investigate impact of sporulation and compost temperatures on feasibility of composting for disposal of carcasses contaminated with Bacillus anthracis. Two strains of B. cereus, 805 and 1391, were sporulated at either 20 or 37°C (Sporulation temperature, ST) and 7 Log10 CFU g(-1) spores added to autoclaved manure in nylon bags (pore size 50 μm) or in sealed vials. Vials and nylon bags were embedded into compost in either a sawdust or manure matrix each containing 16 bovine mortalities (average weight 617 ± 33 kg), retrieved from compost at intervals over 217 days and survival of B. cereus spores assessed. A ST of 20°C decreased spore survival by 1·4 log10 CFU g(-1) (P < 0·05) compared to a 37°C ST. Spore survival was strain dependent. Compost temperatures >55°C reduced spore survival (P < 0·05) and more frequently occurred in the sawdust matrix. Sporulation and compost temperatures were key factors influencing survival of B. cereus spores in mortality compost. Composting may be most appropriate for the disposal of carcasses infected with B. anthracis at ambient temperatures ≤20°C under thermophillic composting conditions (>55°C). © 2015 The Society for Applied Microbiology.

Testing and comparison of the coating materials for immunosensors on QCM

NASA Astrophysics Data System (ADS)

Oztuna, Ali; Nazir, Hasan

2012-06-01

In immunoassay based biosensors development studies polymers, as a matrix, and thiol, amine and aldehyde derivative compounds, as a antibody linker, are to be experimented. Aim of this study is to test amine and acetate functional group containing derivatives in liquid phase in order to develop an antibody immobilization strategy for Quartz Crystal Microbalance (QCM) system. In our study, 4-aminothiophenol (4-AT), carboxylated-PVC (PVC-COOH) and aminated- PVC (PVC-NH2) compared with each other as a coating material. Surface of the coated AT-cut gold crystals were characterized with Fourier Transform Infrared spectrometry (FTIR) and Scanning Electron Microscobe (SEM) and tested in a Bacillus anthracis (GenBank: GQ375871) spores immunoassay model system. Subsequently, a series of SEM micrographs were taken again in order to investigate surface morphology and show the presence of the B. anthracis spores on the sensor surface. When experimental results and SEM images were evaluated together, it was suggested that with the synthesis of PVC like open-chained polymers, containing -NH2 and -SH functional groups, B. anthracis spore detection can be accomplished on QCM without requiring complicated immobilization procedures and expensive preliminary preparations.

Gene expression profiling of human alveolar macrophages infected by B. anthracis spores demonstrates TNF-α and NF-κb are key components of the innate immune response to the pathogen

PubMed Central

2009-01-01

Background Bacillus anthracis, the etiologic agent of anthrax, has recently been used as an agent of bioterrorism. The innate immune system initially appears to contain the pathogen at the site of entry. Because the human alveolar macrophage (HAM) plays a key role in lung innate immune responses, studying the HAM response to B. anthracis is important in understanding the pathogenesis of the pulmonary form of this disease. Methods In this paper, the transcriptional profile of B. anthracis spore-treated HAM was compared with that of mock-infected cells, and differentially expressed genes were identified by Affymetrix microarray analysis. A portion of the results were verified by Luminex protein analysis. Results The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The differentially expressed genes were subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, the Promoter Analysis and Interaction Network Toolset (PAINT) and Oncomine analysis. Among the upregulated genes, we identified a group of chemokine ligand, apoptosis, and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-α, NF-κB and their ligands/receptors. In addition to TNF-α, a broad range of cytokines was induced, and this was confirmed at the level of translation by Luminex multiplex protein analysis. PAINT analysis revealed that many of the genes affected by spores contain the binding site for c-Rel, a member of the NF-κB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. However, many of the genes are poorly annotated, indicating that they represent novel functions. Four of the genes most highly regulated by spores have only previously been associated with head and neck and lung carcinomas. Conclusion The results demonstrate not only that TNF-α and NF-κb are key components of the innate immune response to the pathogen, but also that a large part of the mechanisms by which the alveolar macrophage responds to B. anthracis are still unknown as many of the genes involved are poorly annotated. PMID:19744333

Removal of Bacillus anthracis sterne spore from commercial unpasteurized liquid egg white

USDA-ARS?s Scientific Manuscript database

Thermal pasteurization used by the egg industry for controlling vegetative cells of pathogens is ineffective for destroying endospores. There is a strong need in the agri-industries to develop effective intervention strategies to eliminate the possible bioterrorism threat from spore forming bacteria...

Biological Incident Operations: A Guide for Law Enforcement

DTIC Science & Technology

2004-09-01

organisms. Bacteria can vary in size and shape and some have the capability of forming spores . Spores are much hardier since they are more capable of...unintentional dissemination of a biological agent occurred in the anthrax mailings (October 2001) when anthrax spores cross-contaminated machinery...indicate the presence of Bacillus anthracis (anthrax) and Yersinia pestis (plague). Washington DC emergency personnel responded to the incident. As a

The survivability of Bacillus anthracis (Sterne strain) in processed liquid eggs.

PubMed

Khan, Saeed A; Sung, Kidon; Nawaz, Mohamed S; Cerniglia, Carl E; Tamplin, Mark L; Phillips, Robert W; Kelley, Lynda Collins

2009-04-01

In this study, we investigated the survival and inactivation kinetics of a surrogate strain of Bacillus anthracis (Sterne strain) in whole egg (WE), egg white (EW), sugared egg yolk (YSU), and salted egg yolk (YSA) at low (-20, 0, and 5 degrees C), moderate (15, 20, 25, 30, 35, and 40 degrees C), and high storage temperatures (45, 50, 55, and 60 degrees C). Outgrowth of the spores was measured as lag phase duration (LPD). Replication of vegetative cells was measured in terms of growth rate (GR) and maximum population density (MPD). Spore inactivation was recorded as inactivation rate and percent reduction in viable count. In general, spore viability decreased at low and high temperatures and increased at moderate temperatures. At 0 and 5 degrees C, a 60-100% reduction in spore viability was seen within 2-3 weeks in WE and YSU, 0-30% in YSA, and 50-100% in EW. At -20 degrees C, however, no drop in spore titer was observed in YSU and EW but a 20% drop in titer was seen in YSA and 50% in WE within 2-3 weeks. At high temperatures, WE, EW, and YSA produced a 20-50% drop in the spore titer within 1-4h whereas YSU showed 100% inactivation within 0.75 h. At moderate storage temperatures, as the temperature increased from 15 to 40 degrees C, LPD decreased from 13.5 to 0.75 h and MPD reached 0.27-2.2 x1 0(9) CFU/ml in YSU and WE, respectively. Markedly lower growth was observed in YSA (LPD=24-270 h, MPD=9 x 10(5) CFU/ml) and spores were inactivated completely within 1-6h in EW. The survivability and inactivation data of B. anthracis in liquid egg products reported in this investigation will be helpful in developing risk assessment models on food biosecurity.

Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.

2013-04-29

To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samplesmore » tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.« less

Computational Fluid Dynamics Modeling of Bacillus anthracis ...

EPA Pesticide Factsheets

Journal Article Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. Four different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Despite the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways of the human at the same air concentration of anthrax spores. This greater deposition of spores in the upper airways in the human resulted in lower penetration and deposition in the tracheobronchial airways and the deep lung than that predict

Recombinant protective antigen anthrax vaccine improves survival when administered as a postexposure prophylaxis countermeasure with antibiotic in the New Zealand white rabbit model of inhalation anthrax.

PubMed

Leffel, Elizabeth K; Bourdage, James S; Williamson, E Diane; Duchars, Matthew; Fuerst, Thomas R; Fusco, Peter C

2012-08-01

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 (LD(50)) 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.

Poly(3-hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation.

PubMed

Sadykov, Marat R; Ahn, Jong-Sam; Widhelm, Todd J; Eckrich, Valerie M; Endres, Jennifer L; Driks, Adam; Rutkowski, Gregory E; Wingerd, Kevin L; Bayles, Kenneth W

2017-06-01

Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species. © 2017 John Wiley & Sons Ltd.

Antimicrobial Effects of Gold/Copper Sulphide (Au/Cus) Core/Shell Nanoparticles on Bacillus Anthracis Spores and Cells

DTIC Science & Technology

2013-01-01

namely cutaneous (through skin abrasions or skin lesions caused by biting insects ), gastrointestinal (by the ingestion of spore contaminated food...Journal of Bacteriology, 1939. 37(4): p. 447-460. 42. Hollaender, A. and B.M. Duggar, Irradiation of Plant Viruses and of Microörganisms with

Influence of temperature and organic load on chemical disinfection of Geobacillus steareothermophilus spores, a surrogate for Bacillus anthracis

PubMed Central

Guan, Jiewen; Chan, Maria; Brooks, Brian W.; Rohonczy, Liz

2013-01-01

This study evaluated the influence of temperature and organic load on the effectiveness of domestic bleach (DB), Surface Decontamination Foam (SDF), and Virkon in inactivating Geobacillus stearothermophilus spores, which are a surrogate for Bacillus anthracis spores. The spores were suspended in light or heavy organic preparations and the suspension was applied to stainless steel carrier disks. The dried spore inoculum was covered with the disinfectants and the disks were then incubated at various temperatures. At −20°C, the 3 disinfectants caused less than a 2.0 log10 reduction of spores in both organic preparations during a 24-h test period. At 4°C, the DB caused a 4.4 log10 reduction of spores in light organic preparations within 2 h, which was about 3 log10 higher than what was achieved with SDF or Virkon. In heavy organic preparations, after 24 h at 4°C the SDF had reduced the spore count by 4.5 log10, which was about 2 log10 higher than for DB or Virkon. In general, the disinfectants were most effective at 23°C but a 24-h contact time was required for SDF and Virkon to reduce spore counts in both organic preparations by at least 5.5 log10. Comparable disinfecting activity with DB only occurred with the light organic load. In summary, at temperatures as low as 4°C, DB was the most effective disinfectant, inactivating spores within 2 h on surfaces with a light organic load, whereas SDF produced the greatest reduction of spores within 24 h on surfaces with a heavy organic load. PMID:24082400

Developing a Table of Forces for Human Activity as It Relates ...

EPA Pesticide Factsheets

Report The purpose of this project was to evaluate forces generated by human activity that may cause reaerosolization of Bacillus anthracis spores in the aftermath of an intentional release. Understanding human interaction is important to inform communities on activities that may cause the re-distribution of spores. This report presents the results of an extensive literature review of human-surface interaction forces with respect to Bacillus spore reaerosolization in the outdoor environment.

[Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

PubMed

Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

2015-01-01

To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were <0.001), respectively. The specificity of Plague-UPT-LF and Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA for B.anthracis was 7/16 only. The good performance including rapidness, simplicity and high sensitivity will bring the bright future of UPT-LF to be broadly used on-site as first response to bio-terrorism.

Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kabilan, S.; Suffield, S. R.; Recknagle, K. P.

Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathingmore » conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.« less

Multi-Probe Investigation of Proteomic Structure of Pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malkin, A J; Plomp, M; Leighton, T J

Complete genome sequences are available for understanding biotransformation, environmental resistance and pathogenesis of microbial, cellular and pathogen systems. The present technological and scientific challenges are to unravel the relationships between the organization and function of protein complexes at cell, microbial and pathogens surfaces, to understand how these complexes evolve during the bacterial, cellular and pathogen life cycles, and how they respond to environmental changes, chemical stimulants and therapeutics. In particular, elucidating the molecular structure and architecture of human pathogen surfaces is essential to understanding mechanisms of pathogenesis, immune response, physicochemical interactions, environmental resistance and development of countermeasures against bioterrorist agents.more » The objective of this project was to investigate the architecture, proteomic structure, and function of bacterial spores through a combination of high-resolution in vitro atomic force microscopy (AFM) and AFM-based immunolabeling with threat-specific antibodies. Particular attention in this project was focused on spore forming Bacillus species including the Sterne vaccine strain of Bacillus anthracis and the spore forming near-neighbor of Clostridium botulinum, C. novyi-NT. Bacillus species, including B. anthracis, the causative agent of inhalation anthrax are laboratory models for elucidating spore structure/function. Even though the complete genome sequence is available for B. subtilis, cereus, anthracis and other species, the determination and composition of spore structure/function is not understood. Prof. B. Vogelstein and colleagues at the John Hopkins University have recently developed a breakthrough bacteriolytic therapy for cancer treatment (1). They discovered that intravenously injected Clostridium novyi-NT spores germinate exclusively within the avascular regions of tumors in mice and destroy advanced cancerous lesions. The bacteria were also found to significantly improve the efficacy of chemotherapeutic drugs and radiotherapy (2,3). Currently, there is no understanding of the structure-function relationships of Clostridium novyi-NT spores. As well as their therapeutic interest, studies of Clostridium noyii spores could provide a model for further studies of human pathogenic spore formers including Clostridium botulinum and Clostridium perfringens. This project involved a multi-institutional collaboration of our LLNL group with the groups of Prof. T.J. Leighton (Children's Hospital Oakland Research Institute) and Prof. B. Vogelstein (The Howard Hughes Medical Institute and the Ludwig Center for Cancer Genetics and Therapeutics at The John Hopkins Sidney Kimmel Comprehensive Cancer Center).« less

Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat.

PubMed

Zhou, Bin; Wirsching, Peter; Janda, Kim D

2002-04-16

A naive, human single-chain Fv (scFv) phage-display library was used in bio-panning against live, native spores of Bacillus subtilis IFO 3336 suspended in solution. A direct in vitro panning and enzyme-linked immunosorbent assay-based selection afforded a panel of nine scFv-phage clones of which two, 5B and 7E, were chosen for further study. These two clones differed in their relative specificity and affinity for spores of B. subtilis IFO 3336 vs. a panel of spores from 11 other Bacillus species/strains. A variety of enzyme-linked immunosorbent assay protocols indicated these scFv-phage clones recognized different spore epitopes. Notably, some spore epitopes markedly changed between the free and microtiter-plate immobilized state as revealed by antibody-phage binding. An additional library selection procedure also was examined by constructing a Fab chain-shuffled sublibrary from the nine positive clones and by using a subtractive panning strategy to remove crossreactivity with B. licheniformis 5A24. The Fab-phage clone 52 was improved compared with 5B and was comparable to 7E in binding B. subtilis IFO 3336 vs. B. licheniformis 5A24, yet showed a distinctive crossreactivity pattern with other spores. We also developed a method to directly detect individual spores by using fluorescently labeled antibody-phage. Finally, a variety of "powders" that might be used in deploying spores of B. anthracis were examined for antibody-phage binding. The strategies described provide a foundation to discover human antibodies specific for native spores of B. anthracis that can be developed as diagnostic and therapeutic reagents.

Impedance Measurements Could Accelerate Phage-Based Identification of Bacillus anthracis and Other Bacteria

DTIC Science & Technology

2016-09-01

The Bacillus-inoculated NSM agar plates were incubated at 35°C for at least 48 h until Gram stains revealed the presence of > 90% Bacillus spores in...longer visible in Gram stained samples. Finally, centrifugation was used to remove soluble debris from the preparation and spore concentrations were...minutes post treatment. Gram Stains . Gram stains were used to track the emergence of vegetative Bacillus cells from spores. In this assay, bacterial

Evaluation of the relationship between the Adenosine Triphosphate (ATP) bioluminescence assay and the presence of Bacillus anthracis spores and vegetative cells.

PubMed

Gibbs, Shawn G; Sayles, Harlan; Colbert, Erica M; Hewlett, Angela; Chaika, Oleg; Smith, Philip W

2014-05-28

The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event.

Validation of a Nylon-Flocked-Swab Protocol for Efficient Recovery of Bacterial Spores from Smooth and Rough Surfaces▿

PubMed Central

Probst, Alexander; Facius, Rainer; Wirth, Reinhard; Moissl-Eichinger, Christine

2010-01-01

In order to meet planetary-protection requirements, culturable bacterial spore loads are measured representatively for the total microbial contamination of spacecraft. However, the National Aeronautics and Space Administration's (NASA's) cotton swab protocols for spore load determination have not changed for decades. To determine whether a more efficient alternative was available, a novel swab was evaluated for recovery of different Bacillus atrophaeus spore concentrations on stainless steel and other surfaces. Two protocols for the nylon-flocked swab (NFS) were validated and compared to the present NASA standard protocol. The results indicate that the novel swab protocols recover 3- to 4-fold more (45.4% and 49.0% recovery efficiency) B. atrophaeus spores than the NASA standard method (13.2%). Moreover, the nylon-flocked-swab protocols were superior in recovery efficiency for spores of seven different Bacillus species, including Bacillus anthracis Sterne (recovery efficiency, 20%). The recovery efficiencies for B. atrophaeus spores from different surfaces showed a variation from 5.9 to 62.0%, depending on the roughness of the surface analyzed. Direct inoculation of the swab resulted in a recovery rate of about 80%, consistent with the results of scanning electron micrographs that allowed detailed comparisons of the two swab types. The results of this investigation will significantly contribute to the cleanliness control of future life detection missions and will provide significant improvement in detection of B. anthracis contamination for law enforcement and security efforts. PMID:20543054

Detection of Bacillus spores within 15 minutes by surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Shende, Chetan; Inscore, Frank; Huang, Hermes; Farquharson, Stuart; Sengupta, Atanu

2012-06-01

Since the distribution of Bacillus anthracis causing spores through the US Postal System, there has been a persistent fear that biological warfare agents (BWAs) will be used by terrorists against our military abroad and our civilians at home. Despite the substantial effort to develop BWA analyzers, they remain either too slow, produce high falsealarm rates, lack sensitivity, or cannot be fielded. Consequently there remains a need for a portable analyzer that can overcome these limitations as expressed at the 2011 Biological Weapons Convention. To meet this need we have been developing a sample system that selectively binds BWAs and produce surface-enhanced Raman (SER) spectra using portable Raman spectrometers. Here we describe the use of a short peptide ligand functionalized on silver nanoparticles to selectively capture Bacillus cereus spores (a surrogate of B. anthracis) and their subsequent detection by SER spectroscopy. This technique was used to specifically detect B. cereus spores over closely related species like B. subtilis belonging to the same genus within 15 minutes. Sensitivity of the method was demonstrated by detecting 104 B. cereus spores/mL of water. The technology, once developed should prove invaluable for rapid monitoring of BWAs, which will immensely help first responders and emergency personnel in implementing appropriate counter measures.

Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.

Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditionsmore » using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Genetic Diversity among Bacillus anthracis Soil Isolates at Fine Geographic Scales

PubMed Central

Bader, Douglas E.

2012-01-01

Environmental samples were collected from carcass sites during and after anthrax outbreaks in 2000 and 2001 in the bison (Bison bison) population within Wood Buffalo National Park and the Hook Lake Region north of Wood Buffalo National Park. Bacillus anthracis spores were isolated from these samples and confirmed using phenotypic characterization and real-time PCR. Confirmed B. anthracis isolates were typed using multiple-locus variable-number tandem repeat analysis (MLVA15) and single-nucleotide-repeat analysis (SNRA). B. anthracis isolates split into two clades based on MLVA15, while SNRA allowed some isolates between carcass sites to be distinguished from each other. SNRA polymorphisms were also present within a single carcass site. Some isolates from different carcass sites having the same SNRA type had divergent MLVA types; this finding leads to questions about hierarchical typing methods and the robustness of the fine-scale typing of Bacillus anthracis. PMID:22773624

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

PubMed

Kim, Yeon Hee; Kim, Kyung Ae; Kim, Yu-Ri; Choi, Min Kyung; Kim, Hye Kyeong; Choi, Ki Ju; Chun, Jeong-Hoon; Cha, Kiweon; Hong, Kee-Jong; Lee, Na Gyong; Yoo, Cheon-Kwon; Oh, Hee-Bok; Kim, Tae Sung; Rhie, Gi-eun

2014-01-01

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacillus Anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but not to Macrophages

DTIC Science & Technology

2007-09-01

immunofluorescence (IFM) and light microscopy. Samples were fixed in forma- lin, stained with immunofluorescent dyes (as described below) or spore stain ( malachite ...BEC. Adherence was assessed by microscopic observation of the infected cells stained with malachite green and counterstaining of the BEC. For enzymatic...this significant difference, BEC infected with spores were stained with malachite green and counter- stained with Wright-Giemsa (Fig. 1B and C). This

Effects of L-Alanine and Inosine Germinants on the Elasticity of Bacillus anthracis Spores

DTIC Science & Technology

2010-01-22

there was some scanner hysteresis and drift in the detection system, we often observed that the deflection of the free cantilever was not equal to zero...of waste products.5,35 The degradation of the spore coat resulted in a cell that could bemore easily indented by the AFMprobe, as was observed on...changes in the mechanical properties of the spore. In this work, we use atomic force microscopy (AFM) to characterize the mechanical properties of the

Anthrax lethal and edema toxins in anthrax pathogenesis

PubMed Central

Liu, Shihui; Moayeri, Mahtab; Leppla, Stephen H.

2014-01-01

The pathophysiological effects resulting from many bacterial diseases are caused by exotoxins released by the bacteria. Bacillus anthracis, a spore-forming bacterium, is such a pathogen, causing anthrax through a combination of bacterial infection and toxemia. B. anthracis causes natural infection in humans and animals and has been a top bioterrorism concern since the 2001 anthrax attacks in the USA. The exotoxins secreted by B. anthracis use CMG2 as the major toxin receptor and play essential roles in pathogenesis during the entire course of the disease. This review focuses on the activities of anthrax toxins and their roles in initial and late stages of anthrax infection. PMID:24684968

Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting▿ †

PubMed Central

Leski, Tomasz A.; Caswell, Clayton C.; Pawlowski, Marcin; Klinke, David J.; Bujnicki, Janusz M.; Hart, Sean J.; Lukomski, Slawomir

2009-01-01

The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms. PMID:19767469

A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA, lef and cya genes.

PubMed

Chitlaru, Theodor; Israeli, Ma'ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

2017-10-20

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA<ΔhtrAΔcya<ΔhtrAΔlef<ΔhtrAΔlefΔcya) in attenuation - up to 10 8 -fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (10 9 spores) or double doses (>10 7 spores) of the most attenuated triple mutant strain SterneΔhtrAlef MUT Δcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 10 5 spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30weeks, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Achieving Consistent Multiple Daily Low-Dose Bacillus anthracis Spore Inhalation Exposures in the Rabbit Model

PubMed Central

Barnewall, Roy E.; Comer, Jason E.; Miller, Brian D.; Gutting, Bradford W.; Wolfe, Daniel N.; Director-Myska, Alison E.; Nichols, Tonya L.; Taft, Sarah C.

2012-01-01

Repeated low-level exposures to biological agents could occur before or after the remediation of an environmental release. This is especially true for persistent agents such as B. anthracis spores, the causative agent of anthrax. Studies were conducted to examine aerosol methods needed for consistent daily low aerosol concentrations to deliver a low-dose (less than 106 colony forming units (CFU) of B. anthracis spores) and included a pilot feasibility characterization study, acute exposure study, and a multiple 15 day exposure study. This manuscript focuses on the state-of-the-science aerosol methodologies used to generate and aerosolize consistent daily low aerosol concentrations and resultant low inhalation doses to rabbits. The pilot feasibility characterization study determined that the aerosol system was consistent and capable of producing very low aerosol concentrations. In the acute, single day exposure experiment, targeted inhaled doses of 1 × 102, 1 × 103, 1 × 104, and 1 × 105 CFU were used. In the multiple daily exposure experiment, rabbits were exposed multiple days to targeted inhaled doses of 1 × 102, 1 × 103, and 1 × 104 CFU. In all studies, targeted inhaled doses remained consistent from rabbit-to-rabbit and day-to-day. The aerosol system produced aerosolized spores within the optimal mass median aerodynamic diameter particle size range to reach deep lung alveoli. Consistency of the inhaled dose was aided by monitoring and recording respiratory parameters during the exposure with real-time plethysmography. Overall, the presented results show that the animal aerosol system was stable and highly reproducible between different studies and over multiple exposure days. PMID:22919662

Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen.

PubMed

Henning, Lisa N; Carpenter, Sarah; Stark, Gregory V; Serbina, Natalya V

2018-02-01

The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response. Copyright © 2018 Henning et al.

Inactivation of Bacillus anthracis spores to decontaminate subway railcar and related materials via the fogging of peracetic acid and hydrogen peroxide sporicidal liquids.

PubMed

Richter, William R; Wood, Joseph P; Wendling, Morgan Q S; Rogers, James V

2018-01-15

The inactivation of Bacillus anthracis spores on subway and used subway railcar materials was evaluated using fogged peracetic acid/hydrogen peroxide (PAA) and hydrogen peroxide (H 2 O 2 ). A total of 21 separate decontamination tests were conducted using bacterial spores of both B. anthracis Ames (B.a.) and Bacillus atrophaeus (B.g.) inoculated onto several types of materials. Tests were conducted using commercial off-the-shelf fogging equipment filled with either PAA or H 2 O 2 to fumigate a ∼15 cubic meter chamber under uncontrolled ambient relative humidity and controlled temperature (10 or 20 °C) from 8 to 168 h. For the present study, no conditions were found that resulted in complete inactivation of either B.a. Ames or B.g. on all test materials. Approximately 41% and 38% of the decontamination efficacies for B.a. and B.g., respectively, exhibited ≥6 log 10 reduction (LR); efficacy depended greatly on the material. When testing at 10 °C, the mean LR was consistently lower for both B.a. and B.g. as compared to 20 °C. Based on the statistical comparison of the LR results, B.g. exhibited equivalent or greater resistance than B.a. for approximately 92% of the time across all 21 tests. The efficacy data suggest that B.g. may be a suitable surrogate for B.a. Ames when assessing the decontamination efficacy of fogged PAA or H 2 O 2 . Moreover, the results of this testing indicate that in the event of B.a. spore release into a subway system, the fogging of PAA or H 2 O 2 represents a decontamination option for consideration. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Bacillus anthracis Genome Sequence from the Sverdlovsk 1979 Autopsy Specimens

PubMed Central

Sahl, Jason W.; Pearson, Talima; Okinaka, Richard; Schupp, James M.; Gillece, John D.; Heaton, Hannah; Birdsell, Dawn; Hepp, Crystal; Fofanov, Viacheslav; Noseda, Ramón; Fasanella, Antonio; Hoffmaster, Alex; Wagner, David M.

2016-01-01

ABSTRACT Anthrax is a zoonotic disease that occurs naturally in wild and domestic animals but has been used by both state-sponsored programs and terrorists as a biological weapon. A Soviet industrial production facility in Sverdlovsk, USSR, proved deficient in 1979 when a plume of spores was accidentally released and resulted in one of the largest known human anthrax outbreaks. In order to understand this outbreak and others, we generated a Bacillus anthracis population genetic database based upon whole-genome analysis to identify all single-nucleotide polymorphisms (SNPs) across a reference genome. Phylogenetic analysis has defined three major clades (A, B, and C), B and C being relatively rare compared to A. The A clade has numerous subclades, including a major polytomy named the trans-Eurasian (TEA) group. The TEA radiation is a dominant evolutionary feature of B. anthracis, with many contemporary populations having resulted from a large spatial dispersal of spores from a single source. Two autopsy specimens from the Sverdlovsk outbreak were deep sequenced to produce draft B. anthracis genomes. This allowed the phylogenetic placement of the Sverdlovsk strain into a clade with two Asian live vaccine strains, including the Russian Tsiankovskii strain. The genome was examined for evidence of drug resistance manipulation or other genetic engineering, but none was found. The Soviet Sverdlovsk strain genome is consistent with a wild-type strain from Russia that had no evidence of genetic manipulation during its industrial production. This work provides insights into the world’s largest biological weapons program and provides an extensive B. anthracis phylogenetic reference. PMID:27677796

Chemical and Biological Sensor Standards Study

DTIC Science & Technology

2005-01-01

that is utilized in lieu of Bacillus anthracis in testing biological agent sensors; both are gram positive, spore forming bacteria that have similar...for a given agent dosage is as follows: C = D r 3 f B Tη4π 3 ρ See the table for the variable designation. Using Bacillus anthracis as an example...e.g., genetic similarity, aerosol dynamics, size, shape, etc.) of the agent of interest. For example, Bacillus globigii is a widely used bacterium

Nano-Mechanical Properties of Heat Inactivated Bacillus anthracis and Bacillus thuringiensis Spores

DTIC Science & Technology

2008-03-01

Scanner Laser Mirror Cantilever Sample Probe Tip 16 cereus strain 569, and Bacillus globigii var. niger . Zolock determined that there wer...been used to measure the surface elasticities of a variety of microbial organisms including Pseudomonas putida, Bacillus subtilis, Aspergillus ...66:307-311 (2005). Zhao, Liming, David Schaefer, and Mark R. Marten. “Assessment of Elasticity and Topography of Aspergillus nidulans Spores via

Vaporous Decontamination Methods: Potential Uses and Research Priorities for Chemical and Biological Contamination Control

DTIC Science & Technology

2006-06-01

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen...resistant to commonly used disinfectants and require the use of chemical sterilants † to effectively decontaminate exposed areas. Since anthrax...spores can aerosolise the use of vaporous sterilants in the remediation of contaminated areas is desirable. A number of vaporous sterilants exist which

Capture of Aerosols by Iodinated Fiber Media

DTIC Science & Technology

2004-09-15

fibrous media if provided with 70-80% relative humidity and atmospheric dust (Maus et al., 2000). Spore -forming bacteria such as Bacillus anthracis are...States. The anthrax spores sent out during these attacks were classified as being highly concentrated and processed to be disseminated and inhaled...media, and produce more undesirable bioaerosols. This phenomenon has been reported in many studies in heating, ventilation, and air conditioning ( HVAC

75 FR 66085 - Agency Information Collection Activities; Proposed Collection; Comment Request; Certification of...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-27

... comply with the terms and conditions of the pesticide registration (e.g., registrants of anthrax-related pesticide products that assert claims to inactivate bacillus anthracis (anthrax) spores). Paperwork...

Evaluation of the House Fly Musca domestica as a Mechanical Vector for an Anthrax

PubMed Central

Fasanella, Antonio; Scasciamacchia, Silvia; Garofolo, Giuliano; Giangaspero, Annunziata; Tarsitano, Elvira; Adone, Rosanna

2010-01-01

Anthrax is a disease of human beings and animals caused by the encapsulated, spore-forming, Bacillus anthracis. The potential role of insects in the spread of B. anthracis to humans and domestic animals during an anthrax outbreak has been confirmed by many studies. Among insect vectors, the house fly Musca domestica is considered a potential agent for disease transmission. In this study, laboratory-bred specimens of Musca domestica were infected by feeding on anthrax-infected rabbit carcass or anthrax contaminated blood, and the presence of anthrax spores in their spots (faeces and vomitus) was microbiologically monitored. It was also evaluated if the anthrax spores were able to germinate and replicate in the gut content of insects. These results confirmed the role of insects in spreading anthrax infection. This role, although not major, given the huge size of fly populations often associated with anthrax epidemics in domestic animals, cannot be neglected from an epidemiological point of view and suggest that fly control should be considered as part of anthrax control programs. PMID:20808920

Data for methyl bromide decon testing

EPA Pesticide Factsheets

Spreadsheets containing data for recovery of spores from different materials. Data on the fumigation parameters are also included.This dataset is associated with the following publication:Wood , J., M. Wendling, W. Richter, A. Lastivka, and L. Mickelsen. Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, USA, 1-28, (2016).

Comparative Evaluation of Vacuum-based Surface Sampling ...

EPA Pesticide Factsheets

Journal Article Following a biological contamination incident, collection of surface samples is necessary to determine the extent and level of contamination, and to deem an area safe for reentry upon decontamination. Current sampling strategies targeting Bacillus anthracis spores prescribe vacuum-based methods for rough and/or porous surfaces. In this study, four commonly-used B. anthracis spore sampling devices (vacuum socks, 37 mm 0.8 µm MCE filter cassettes, 37 mm 0.3 µm PTFE filter cassettes, and 3MTM forensic filters) were comparatively evaluated for their ability to recover surface-associated spores. The vacuum sock device was evaluated at two sampling speeds (slow and fast), resulting in five total methods evaluated. Aerosolized spores (~105 cm-2) of a surrogate Bacillus species (Bacillus atrophaeus) were allowed to settle onto three material types (concrete, carpet, and upholstery). Ten replicate samples were collected using each vacuum method, from each of the three material types. In addition, stainless steel (i.e., nonporous) surfaces inoculated simultaneously were sampled with pre-moistened wipes. Recoveries from wipes of steel surfaces were utilized to verify the inoculum, and to normalize vacuum-based recoveries across trials. Recovery (CFU cm-2) and relative recovery (vacuum recovery/wipe recovery) were determined for each method and material type. Relative recoveries were compared by one-way and three-way ANOVA. Data analysis by one-

Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station.

PubMed

Venkateswaran, Kasthuri; Singh, Nitin K; Checinska Sielaff, Aleksandra; Pope, Robert K; Bergman, Nicholas H; van Tongeren, Sandra P; Patel, Nisha B; Lawson, Paul A; Satomi, Masataka; Williamson, Charles H D; Sahl, Jason W; Keim, Paul; Pierson, Duane; Perry, Jay

2017-01-01

In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis - B. cereus - B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis . Whole-genome sequence characterizations showed that ISS strains had the plcR non- B. anthracis ancestral "C" allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis . The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis . Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis . IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station's surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic ( B. anthracis ), food poisoning ( B. cereus ), and biotechnologically useful ( B. thuringiensis ) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.

Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station

PubMed Central

Singh, Nitin K.; Checinska Sielaff, Aleksandra; Pope, Robert K.; Bergman, Nicholas H.; van Tongeren, Sandra P.; Patel, Nisha B.; Lawson, Paul A.; Satomi, Masataka; Williamson, Charles H. D.; Sahl, Jason W.; Pierson, Duane; Perry, Jay

2017-01-01

ABSTRACT In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization–time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral “C” allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station’s surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions. PMID:28680972

Bacillus anthracis (image)

MedlinePlus

... aerobic spore-forming bacterium that causes disease in humans and animals. The bacteria is found in two forms: cutaneous anthrax and inhalation anthrax. Cutaneous anthrax is an infection of the skin caused by direct contact with the bacterium. Inhalation ...

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

PubMed

Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

2016-01-01

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh

PubMed Central

Rume, Farzana Islam; Affuso, Alessia; Serrecchia, Luigina; Rondinone, Valeria; Manzulli, Viviana; Campese, Emanuele; Di Taranto, Pietro; Biswas, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Yasmin, Mahmuda; Fasanella, Antonio; Hugh-Jones, Martin

2016-01-01

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country. PMID:27082248

Differences in susceptibility of inbred mice to Bacillus anthracis.

PubMed Central

Welkos, S L; Keener, T J; Gibbs, P H

1986-01-01

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. PMID:3081444

Quantitative Method To Determine Sporicidal Decontamination of Building Surfaces by Gaseous Fumigants, and Issues Related to Laboratory-Scale Studies▿

PubMed Central

Rastogi, Vipin K.; Wallace, Lalena; Smith, Lisa S.; Ryan, Shawn P.; Martin, Blair

2009-01-01

Chlorine dioxide gas and vaporous hydrogen peroxide sterilant have been used in the cleanup of building interiors contaminated with spores of Bacillus anthracis. A systematic study, in collaboration with the U.S. Environmental Protection Agency, was jointly undertaken by the U.S. Army-Edgewood Chemical Biological Center to determine the sporicidal efficacies of these two fumigants on six building structural materials: carpet, ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. Critical issues related to high-throughput sample processing and spore recovery from porous and nonporous surfaces included (i) the extraction of spores from complex building materials, (ii) the effects of titer challenge levels on fumigant efficacy, and (iii) the impact of bioburden inclusion on spore recovery from surfaces and spore inactivation. Small pieces (1.3 by 1.3 cm of carpet, ceiling tile, wallboard, I-beam steel, and pinewood and 2.5 by 1.3 cm for cinder block) of the materials were inoculated with an aliquot of 50 μl containing the target number (1 × 106, 1 × 107, or 1 × 108) of avirulent spores of B. anthracis NNR1Δ1. The aliquot was dried overnight in a biosafety cabinet, and the spores were extracted by a combination of a 10-min sonication and a 2-min vortexing using 0.5% buffered peptone water as the recovery medium. No statistically significant drop in the kill efficacies of the fumigants was observed when the spore challenge level was increased from 6 log units to 8 log units, even though a general trend toward inhibition of fumigant efficacy was evident. The organic burden (0 to 5%) in the spore inoculum resulted in a statistically significant drop in spore recovery (at the 2 or 5% level). The effect on spore killing was a function of the organic bioburden amount and the material type. In summary, a high-throughput quantitative method was developed for determining the efficacies of fumigants, and the spore recoveries from five porous materials and one nonporous material ranged between 20 and 80%. PMID:19346341

Quantitative method to determine sporicidal decontamination of building surfaces by gaseous fumigants, and issues related to laboratory-scale studies.

PubMed

Rastogi, Vipin K; Wallace, Lalena; Smith, Lisa S; Ryan, Shawn P; Martin, Blair

2009-06-01

Chlorine dioxide gas and vaporous hydrogen peroxide sterilant have been used in the cleanup of building interiors contaminated with spores of Bacillus anthracis. A systematic study, in collaboration with the U.S. Environmental Protection Agency, was jointly undertaken by the U.S. Army-Edgewood Chemical Biological Center to determine the sporicidal efficacies of these two fumigants on six building structural materials: carpet, ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. Critical issues related to high-throughput sample processing and spore recovery from porous and nonporous surfaces included (i) the extraction of spores from complex building materials, (ii) the effects of titer challenge levels on fumigant efficacy, and (iii) the impact of bioburden inclusion on spore recovery from surfaces and spore inactivation. Small pieces (1.3 by 1.3 cm of carpet, ceiling tile, wallboard, I-beam steel, and pinewood and 2.5 by 1.3 cm for cinder block) of the materials were inoculated with an aliquot of 50 microl containing the target number (1 x 10(6), 1 x 10(7), or 1 x 10(8)) of avirulent spores of B. anthracis NNR1Delta1. The aliquot was dried overnight in a biosafety cabinet, and the spores were extracted by a combination of a 10-min sonication and a 2-min vortexing using 0.5% buffered peptone water as the recovery medium. No statistically significant drop in the kill efficacies of the fumigants was observed when the spore challenge level was increased from 6 log units to 8 log units, even though a general trend toward inhibition of fumigant efficacy was evident. The organic burden (0 to 5%) in the spore inoculum resulted in a statistically significant drop in spore recovery (at the 2 or 5% level). The effect on spore killing was a function of the organic bioburden amount and the material type. In summary, a high-throughput quantitative method was developed for determining the efficacies of fumigants, and the spore recoveries from five porous materials and one nonporous material ranged between 20 and 80%.

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis.

PubMed

Makam, Shivakiran S; Majumder, Saugata; Kingston, Joseph J; Urs, Radhika M; Tuteja, Urmil; Sripathi, Murali H; Batra, Harsh V

2013-12-01

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml⁻¹ of bacterial cells and spores, respectively. IPCR was found to be 2-3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Systematic Evaluation of Aggressive Air Sampling for Bacillus ...

EPA Pesticide Factsheets

Report The primary objectives of this project were to evaluate the Aggressive Air Sampling (AAS) method compared to currently used surface sampling methods and to determine if AAS is a viable option for sampling Bacillus anthracis spores.

An adenovirus-vectored nasal vaccine confers rapid and sustained protection against anthrax in a single-dose regimen.

PubMed

Zhang, Jianfeng; Jex, Edward; Feng, Tsungwei; Sivko, Gloria S; Baillie, Leslie W; Goldman, Stanley; Van Kampen, Kent R; Tang, De-chu C

2013-01-01

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine.

An Adenovirus-Vectored Nasal Vaccine Confers Rapid and Sustained Protection against Anthrax in a Single-Dose Regimen

PubMed Central

Jex, Edward; Feng, Tsungwei; Sivko, Gloria S.; Baillie, Leslie W.; Goldman, Stanley; Van Kampen, Kent R.; Tang, De-chu C.

2013-01-01

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine. PMID:23100479

[An historical, sociocultural view and in the fiction literature of Bacillus anthracis cases by shaving brushes].

PubMed

Vázquez-Espinosa, E; Laganà, C; Vazquez, F

2018-06-01

In the period from 1915 to 1924 anthrax outbreaks were described by Bacillus anthracis due to the contamination of razor brushes that reached Europe and the United States from areas such as Japan, China or Russia. The brushes were made with badger hair, and then, to reduce the cost with horse hair and other animals. World War I supoosed that the traffics of these brushes, that passed through Europe, changed and the processes of sterilization of the same were deficient giving rise to these outbreaks, that in a percentage of 20% produced the death of the users. The impact of the fashion of wearing a beard, the presence of these cases in the press, in the society of that period, and literature are studied through the work of Agatha Christie who wrote, in 1936, the Hercules Poirot´s novel Cards on the table, and where she describes the murder of one of the characters with the shaving brush contaminated with Bacillus anthracis spores. ©The Author 2018. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).

Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination

DTIC Science & Technology

2009-12-01

exosporium maturation and assembly and suggest a novel role for the exosporium in germination. During starvation, bacteria of the genus Bacillus...Bacillus subtilis, the outermost struc- ture is a protective layer called the coat, which guards the spore against reactive small molecules, degradative ...analysis. Generation of anti-ExsK antibodies. Recombinant ExsK was generated and purified using the pET expression system (Novagen) according to the

Morphogenesis of the Bacillus anthracis Spore

DTIC Science & Technology

2007-02-01

product and plasmid pEO-3 (53) with BamHI and HindIII and ligated the resulting DNA fragments to build pRG23. We passaged pRG23 through E. coli GM1684...to remove debris. The removal of the exosporium was confirmed by elec- tron microscopy (data not shown). Antibody production . To produce a...O. Henriques, and S. M. Cutting (ed.), Bacterial spore formers: probiotics and emerging applications. Horizon Bioscience, Norfolk, United Kingdom

Dendritic Cells Endocytose Bacillus Anthracis Spores: Implications for Anthrax Pathogenesis

DTIC Science & Technology

2005-02-15

29. Horwitz, M. A. 1984. Phagocytosis of the Legionnaires ’ disease bacterium (Le- gionella pneumophila) occurs by a novel mechanism: engulfment within...Journal of Immunology, 2005, 174: 5545–5552. I nhalational anthrax, a disease that was exploited for bioter-rorism (1), is most often fatal and causes...them out of the lungs. However, mice that were chemically depleted of macrophages and infected with spores by aerosol nev- ertheless experienced disease

Anthrax Lethal Toxin Impairs Innate Immune Functions of Alveolar Macrophages and Facilitates Bacillus anthracis Survival

PubMed Central

Ribot, Wilson J.; Panchal, Rekha G.; Brittingham, Katherine C.; Ruthel, Gordon; Kenny, Tara A.; Lane, Douglas; Curry, Bob; Hoover, Timothy A.; Friedlander, Arthur M.; Bavari, Sina

2006-01-01

Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses. Although several factors contribute to inhalational anthrax, we hypothesized that unimpeded infection of Bacillus anthracis is directly linked to disabling the innate immune functions contributed by AM. Here, we investigated the effects of lethal toxin (LT), one of the binary complex virulence factors produced by B. anthracis, on freshly isolated nonhuman primate AM. Exposure of AM to doses of LT that killed susceptible macrophages had no effect on the viability of AM, despite complete MEK1 cleavage. Intoxicated AM remained fully capable of B. anthracis spore phagocytosis. However, pretreatment of AM with LT resulted in a significant decrease in the clearance of both the Sterne strain and the fully virulent Ames strain of B. anthracis, which may have been a result of impaired AM secretion of proinflammatory cytokines. Our data imply that cytolysis does not correlate with MEK1 cleavage, and this is the first report of LT-mediated impairment of nonhuman primate AM bactericidal activity against B. anthracis. PMID:16926394

Immunological correlates for protection against intranasal challenge of Bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits.

PubMed

Weiss, Shay; Kobiler, David; Levy, Haim; Marcus, Hadar; Pass, Avi; Rothschild, Nili; Altboum, Zeev

2006-01-01

Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 x 10(5) conferred protection, whereas lower titers (between 10(4) and 10(5)) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.

Operational Testing of Floor Cleaning Cloths for Household ...

EPA Pesticide Factsheets

Report The objective of this study was to evaluate the Swiffer® Sweeper® floor mop system (SSFMS) as a low-tech method to clean indoor residential floors contaminated with B. anthracis spores (the causative agent of anthrax).

LABORATORY GUIDELINES FOR ANALYSIS OF BIOTERRORISM SAMPLES

EPA Science Inventory

With advent of deaths associated with Bacillus anthracis spore contaminated mail, a worldwide need was apparent for increased laboratory capacity to safely analyze bioterrorism samples. The U.S. Department of Health and Human Services has furnished guidelines for microbiological...

Peptide Conjugated Phosphorodiamidate Morpholino Oligomers Increase Survival of Mice Challenged with Ames Bacillus anthracis

PubMed Central

Geller, Bruce L.; Mellbye, Brett; Lane, Douglas; Iversen, Patrick L.; Bavari, Sina

2012-01-01

Targeting bacterial essential genes using antisense phosphorodiamidate morpholino oligomers (PMOs) represents an important strategy in the development of novel antibacterial therapeutics. PMOs are neutral DNA analogues that inhibit gene expression in a sequence-specific manner. In this study, several cationic, membrane-penetrating peptides were conjugated to PMOs (PPMOs) that target 2 bacterial essential genes: acyl carrier protein (acpP) and gyrase A (gyrA). These were tested for their ability to inhibit growth of Bacillus anthracis, a gram-positive spore-forming bacterium and causative agent of anthrax. PPMOs targeted upstream of both target gene start codons and conjugated with the bacterium-permeating peptide (RFF)3R were found to be most effective in inhibiting bacterial growth in vitro. Both of the gene-targeted PPMOs protected macrophages from B. anthracis induced cell death. Subsequent, in vivo testing of the PPMOs resulted in increased survival of mice challenged with the virulent Ames strain of B. anthracis. Together, these studies suggest that PPMOs targeting essential genes have the potential of being used as antisense antibiotics to treat B. anthracis infections. PMID:22978365

Holographic deep learning for rapid optical screening of anthrax spores

PubMed Central

Jo, YoungJu; Park, Sangjin; Jung, JaeHwang; Yoon, Jonghee; Joo, Hosung; Kim, Min-hyeok; Kang, Suk-Jo; Choi, Myung Chul; Lee, Sang Yup; Park, YongKeun

2017-01-01

Establishing early warning systems for anthrax attacks is crucial in biodefense. Despite numerous studies for decades, the limited sensitivity of conventional biochemical methods essentially requires preprocessing steps and thus has limitations to be used in realistic settings of biological warfare. We present an optical method for rapid and label-free screening of Bacillus anthracis spores through the synergistic application of holographic microscopy and deep learning. A deep convolutional neural network is designed to classify holographic images of unlabeled living cells. After training, the network outperforms previous techniques in all accuracy measures, achieving single-spore sensitivity and subgenus specificity. The unique “representation learning” capability of deep learning enables direct training from raw images instead of manually extracted features. The method automatically recognizes key biological traits encoded in the images and exploits them as fingerprints. This remarkable learning ability makes the proposed method readily applicable to classifying various single cells in addition to B. anthracis, as demonstrated for the diagnosis of Listeria monocytogenes, without any modification. We believe that our strategy will make holographic microscopy more accessible to medical doctors and biomedical scientists for easy, rapid, and accurate point-of-care diagnosis of pathogens. PMID:28798957

Ecological suitability modeling for anthrax in the Kruger National Park, South Africa.

PubMed

Steenkamp, Pieter Johan; van Heerden, Henriette; van Schalkwyk, Ockert Louis

2018-01-01

The spores of the soil-borne bacterium, Bacillus anthracis, which causes anthrax are highly resistant to adverse environmental conditions. Under ideal conditions, anthrax spores can survive for many years in the soil. Anthrax is known to be endemic in the northern part of Kruger National Park (KNP) in South Africa (SA), with occasional epidemics spreading southward. The aim of this study was to identify and map areas that are ecologically suitable for the harboring of B. anthracis spores within the KNP. Anthrax surveillance data and selected environmental variables were used as inputs to the maximum entropy (Maxent) species distribution modeling method. Anthrax positive carcasses from 1988-2011 in KNP (n = 597) and a total of 40 environmental variables were used to predict and evaluate their relative contribution to suitability for anthrax occurrence in KNP. The environmental variables that contributed the most to the occurrence of anthrax were soil type, normalized difference vegetation index (NDVI) and precipitation. Apart from the endemic Pafuri region, several other areas within KNP were classified as ecologically suitable. The outputs of this study could guide future surveillance efforts to focus on predicted suitable areas for anthrax, since the KNP currently uses passive surveillance to detect anthrax outbreaks.

Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

PubMed Central

Walper, Scott A.; Anderson, George P.; Brozozog Lee, P. Audrey; Glaven, Richard H.; Liu, Jinny L.; Bernstein, Rachel D.; Zabetakis, Dan; Johnson, Linwood; Czarnecki, Jill M.; Goldman, Ellen R.

2012-01-01

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors. PMID:22412927

Use of the mice passive protection test to evaluate the humoral response in goats vaccinated with Sterne 34F2 live spore vaccine.

PubMed

Phaswana, P H; Ndumnego, O C; Koehler, S M; Beyer, W; Crafford, J E; van Heerden, H

2017-09-07

The Sterne live spore vaccine (34F2) is the most widely used veterinary vaccine against anthrax in animals. Antibody responses to several antigens of Bacillus anthracis have been described with a large focus on those against protective antigen (PA). The focus of this study was to evaluate the protective humoral immune response induced by the live spore anthrax vaccine in goats. Boer goats vaccinated twice (week 0 and week 12) with the Sterne live spore vaccine and naive goats were used to monitor the anti-PA and toxin neutralizing antibodies at week 4 and week 17 (after the second vaccine dose) post vaccination. A/J mice were passively immunized with different dilutions of sera from immune and naive goats and then challenged with spores of B. anthracis strain 34F2 to determine the protective capacity of the goat sera. The goat anti-PA ELISA titres indicated significant sero-conversion at week 17 after the second doses of vaccine (p = 0.009). Mice receiving undiluted sera from goats given two doses of vaccine (twice immunized) showed the highest protection (86%) with only 20% of mice receiving 1:1000 diluted sera surviving lethal challenge. The in vitro toxin neutralization assay (TNA) titres correlated to protection of passively immunized A/J mice against lethal infection with the vaccine strain Sterne 34F2 spores using immune goat sera up to a 1:10 dilution (r s  ≥ 0.522, p = 0.046). This study suggests that the passive mouse protection model could be potentially used to evaluate the protective immune response in livestock animals vaccinated with the current live vaccine and new vaccines.

LABORATORY GUIDELINES FOR ANALYSIS OF BIOTERRORISM SAMPLES

EPA Science Inventory

After the attack on the World Trade Center on September 11, 2002, and the subsequent deaths associated with Bacillus anthracis spore contaminated mail, a worldwide need was apparent for increased laboratory capacity to safely analyze bioterrorism samples. The U.S. Department o...

Lethal factor is not required for Bacillus anthracis virulence in guinea pigs and rabbits.

PubMed

Levy, Haim; Weiss, Shay; Altboum, Zeev; Schlomovitz, Josef; Rothschild, Nili; Blachinsky, Eran; Kobiler, David

2011-11-01

The major virulence factor of Bacillus anthracis is the tripartite anthrax toxin, comprising the protective antigen (PA), lethal factor (LF) and edema factor (EF). The LF of B. anthracis is a metalloprotease that has been shown to play an important role in pathogenicity. Deletion of this gene (lef) in the Sterne strain was reported to dramatically reduce the pathogenicity of this strain in mice, and was reported to be as dramatic as the deletion of PA. We evaluated the effect on pathogenicity of the lef deletion in the fully virulent Vollum strain in guinea pigs and NZW rabbits by either subcutaneous injection or intranasal instillation. In guinea pigs, no major differences between the mutant strain and the wild type could be detected in the LD(50) or mean time to death values. On the other hand, the lef deletion caused death of 50-70% of all rabbits infected with the mutant spores at doses equivalent or higher than the wild type LD(50). The surviving rabbits, which were infected with spore doses higher than the wild type LD(50), developed a protective immune response that conferred resistance to challenge with the wild type strain. These findings may indicate that the mutant lacking the LF is capable of host colonization which causes death in 50-70% of the animals and a protective immune response in the others. These results indicate that unlike the data obtained in mice, the LF mutation does not abolish B. anthracis pathogenicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Chlorine Dioxide Fumigation of Subway Materials ...

EPA Pesticide Factsheets

Report This bench scale study observed that a six (6) log reduction in viable spores of a suitable B. anthracis surrogate can be obtained for subway infrastructure materials by ClO2 fumigation if the temperature is at or above 24 °C combined with RH greater than 75%. No six log reduction in viable spores was observed at realistic (winter) temperatures in a subway environment (11-13 °C and 70-80% RH) for periods of fumigation that are otherwise efficacious at 24 °C/ 75% RH.

Efficacy and Immunogenicity of Single-Dose AdVAV Intranasal Anthrax Vaccine Compared to Anthrax Vaccine Absorbed in an Aerosolized Spore Rabbit Challenge Model

PubMed Central

Krishnan, Vyjayanthi; Andersen, Bo H.; Shoemaker, Christine; Sivko, Gloria S.; Tordoff, Kevin P.; Stark, Gregory V.; Zhang, Jianfeng; Feng, Tsungwei; Duchars, Matthew

2015-01-01

AdVAV is a replication-deficient adenovirus type 5-vectored vaccine expressing the 83-kDa protective antigen (PA83) from Bacillus anthracis that is being developed for the prevention of disease caused by inhalation of aerosolized B. anthracis spores. A noninferiority study comparing the efficacy of AdVAV to the currently licensed Anthrax Vaccine Absorbed (AVA; BioThrax) was performed in New Zealand White rabbits using postchallenge survival as the study endpoint (20% noninferiority margin for survival). Three groups of 32 rabbits were vaccinated with a single intranasal dose of AdVAV (7.5 × 107, 1.5 × 109, or 3.5 × 1010 viral particles). Three additional groups of 32 animals received two doses of either intranasal AdVAV (3.5 × 1010 viral particles) or intramuscular AVA (diluted 1:16 or 1:64) 28 days apart. The placebo group of 16 rabbits received a single intranasal dose of AdVAV formulation buffer. All animals were challenged via the inhalation route with a targeted dose of 200 times the 50% lethal dose (LD50) of aerosolized B. anthracis Ames spores 70 days after the initial vaccination and were followed for 3 weeks. PA83 immunogenicity was evaluated by validated toxin neutralizing antibody and serum anti-PA83 IgG enzyme-linked immunosorbent assays (ELISAs). All animals in the placebo cohort died from the challenge. Three of the four AdVAV dose cohorts tested, including two single-dose cohorts, achieved statistical noninferiority relative to the AVA comparator group, with survival rates between 97% and 100%. Vaccination with AdVAV also produced antibody titers with earlier onset and greater persistence than vaccination with AVA. PMID:25673303

A Field Investigation of Bacillus anthracis Contamination of U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October 2001

PubMed Central

Higgins, James A.; Cooper, Mary; Schroeder-Tucker, Linda; Black, Scott; Miller, David; Karns, Jeffrey S.; Manthey, Erlynn; Breeze, Roger; Perdue, Michael L.

2003-01-01

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates. PMID:12514046

INACTIVATION OF BACILLUS GLOBIGII BY CHLORINATION: A HIERARCHICAL BAYESIAN MODEL

EPA Science Inventory

Recent events where spores of Bacillus anthracis have been used as a bioterrorist weapon have prompted interest in determining the resistance of this organism to commonly used disinfectants, such as chlorine, chlorine dioxide and ozone. This work was undertaken to study ...

THE ESTABLISHMENT OF LABORATORY GUIDELINES FOR ANALYSIS OF BIOTERRORISM SAMPLES

EPA Science Inventory

After the attack on the World Trade Center on September 11, 2002, and the subsequent deaths associated with Bacillus anthracis spore contaminated mail, a worldwide need was apparent for increased laboratory capacity to safely analyze bioterrorism samples. The U.S. Department of ...

Nosocomial infection of Serratia marcescens may induce a protective effect in monkeys exposed to Bacillus anthracis.

PubMed

Leffel, Elizabeth K; Twenhafel, Nancy A; Whitehouse, Chris A

2008-08-01

This study was originally designed to collect data on the natural history of inhalational anthrax in a new nonhuman primate model. An uncontrollable event created a new experimental condition which allowed us to retrospectively evaluate the power of the innate immune system to protect from an aerosol exposure of B. anthracis. Five African green monkeys (AGMs) had intravenous catheters implanted. One catheter was accidentally pulled out, leaving four AGMs with catheters and one without. All were exposed, to multiple lethal doses of B. anthracis Ames strain. Blood was collected twice daily to evaluate bacteremia. The AGM with no catheter had blood drawn from a femoral vein and became bacteremic on Day 9; succumbed to inhalational anthrax on Day 10. The other four AGMs had S. marcescens contamination in the catheter; indicated by pure colonies grown from the blood. None of these AGMs showed clinical signs of illness, had B. anthracis or a detectable level of protective antigen in the bloodstream. It appears that the presence of S. marcescens may have induced a "Coley's toxin" effect in this experiment. The innate immune response may have protected the AGMs from a lethal inhalational dose of B. anthracis spores.

Pathogenic ecology: Where have all the pathogens gone? Anthrax: a classic case

NASA Astrophysics Data System (ADS)

Kiel, Johnathan; Walker, Wes W.; Andrews, Carrie J.; De Los Santos, Amy; Adams, Roy N.; Bucholz, Matthew W.; McBurnett, Shelly D.; Fuentes, Vladimir; Rizner, Karon E.; Blount, Keith W.

2009-05-01

Pathogenic ecology is the natural relationship to animate and inanimate components of the environment that support the sustainment of a pathogen in the environment or prohibit its sustainment, or their interactions with an introduced pathogen that allow for the establishment of disease in a new environment. The anthrax bacterium in the spore form has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to determine the environmental reservoir of Bacillus anthracis between outbreaks of anthrax and to examine the potential factors influencing the conversion of the Bacillus anthracis from a quiescent state to the disease causing state. Here we provide environmental and laboratory data for the cycling of Bacillus anthracis in plants to reconcile observations that contradict the soil borne hypothesis of anthrax maintenance in the environment.

Development of an Aerosol Surface Inoculation Method for Bacillus Spores ▿

PubMed Central

Lee, Sang Don; Ryan, Shawn P.; Snyder, Emily Gibb

2011-01-01

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 107 CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies. PMID:21193670

Development of an aerosol surface inoculation method for bacillus spores.

PubMed

Lee, Sang Don; Ryan, Shawn P; Snyder, Emily Gibb

2011-03-01

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.

Anthrax vaccine antigen-adjuvant formulations completely protect New Zealand white rabbits against challenge with Bacillus anthracis Ames strain spores.

PubMed

Peachman, Kristina K; Li, Qin; Matyas, Gary R; Shivachandra, Sathish B; Lovchik, Julie; Lyons, Rick C; Alving, Carl R; Rao, Venigalla B; Rao, Mangala

2012-01-01

In an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model using Bacillus anthracis Ames strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-water emulsion, PA displayed on bacteriophage T4 by the intramuscular route, and PA mixed with Escherichia coli heat-labile enterotoxin administered by the needle-free transcutaneous route. Three of the vaccine formulations administered by the intramuscular or the transcutaneous route as a three-dose regimen induced 100% protection in the rabbit model. One of the formulations, liposomal PA, also induced significantly higher lethal toxin neutralizing antibodies than PA-Alhydrogel. Even 5 months after the second immunization of a two-dose regimen, rabbits vaccinated with liposomal PA were 100% protected from lethal challenge with Ames strain spores. In summary, the needle-free skin delivery and liposomal formulation that were found to be effective in two different animal model systems appear to be promising candidates for next-generation anthrax vaccine development.

Detection of Agar, by Analysis of Sugar Markers, Associated with Bacillus Anthracis Spores, After Culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wunschel, David S.; Colburn, Heather A.; Fox, Alvin

2008-08-01

Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-L-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived frommore » agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artefactual background (reductive hydrolysis) or marker destruction (hydrolysis) lead to the search for alternative sugar markers. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.« less

Ecological suitability modeling for anthrax in the Kruger National Park, South Africa

PubMed Central

Steenkamp, Pieter Johan; van Schalkwyk, Ockert Louis

2018-01-01

The spores of the soil-borne bacterium, Bacillus anthracis, which causes anthrax are highly resistant to adverse environmental conditions. Under ideal conditions, anthrax spores can survive for many years in the soil. Anthrax is known to be endemic in the northern part of Kruger National Park (KNP) in South Africa (SA), with occasional epidemics spreading southward. The aim of this study was to identify and map areas that are ecologically suitable for the harboring of B. anthracis spores within the KNP. Anthrax surveillance data and selected environmental variables were used as inputs to the maximum entropy (Maxent) species distribution modeling method. Anthrax positive carcasses from 1988–2011 in KNP (n = 597) and a total of 40 environmental variables were used to predict and evaluate their relative contribution to suitability for anthrax occurrence in KNP. The environmental variables that contributed the most to the occurrence of anthrax were soil type, normalized difference vegetation index (NDVI) and precipitation. Apart from the endemic Pafuri region, several other areas within KNP were classified as ecologically suitable. The outputs of this study could guide future surveillance efforts to focus on predicted suitable areas for anthrax, since the KNP currently uses passive surveillance to detect anthrax outbreaks. PMID:29377918

Micro-Etched Platforms for Thermal Inactivation of Bacillus Anthracis and Bacillus Thuringiensis Spores

DTIC Science & Technology

2008-03-01

slips was first coated with a detergent wash. Commercially available Ivory soap shavings were diluted with sterile Millipore® water in a...environments. This removed controllable variability between the Bacillus species and increased the confidence in continued use of such surrogacy

Advanced Catalytic Enzyme System (ACES) - Dual Use Capabilities

DTIC Science & Technology

2003-07-01

Novozymes A/S (Bagsvaerd, Denmark) with activity against both cells and spores, and a bacteriophage enzyme (PlyG Lysin) that is specific for B. anthracis... Novozymes ), Lysozyme (commercial) Buffer: Ammonium carbonate Fire-Fighting Components: ColdFire®, Fire Choke® or an equivalent Class A foam The

Mapping of Proteomic Composition on the Surfaces of Bacillus spores by Atomic Force Microscopy-based Immunolabeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plomp, M; Malkin, A J

2008-06-02

Atomic force microscopy provides a unique capability to image high-resolution architecture and structural dynamics of pathogens (e.g. viruses, bacteria and bacterial spores) at near molecular resolution in native conditions. Further development of atomic force microscopy in order to enable the correlation of pathogen protein surface structures with specific gene products is essential to understand the mechanisms of the pathogen life cycle. We have applied an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures through the visualization of the binding of antibodies, conjugated with nanogold particles, to specific epitopes on Bacillus spore surfaces. This information is generated while simultaneouslymore » acquiring the surface morphology of the pathogen. The immunospecificity of this labeling method was established through the utilization of specific polyclonal and monoclonal antibodies that target spore coat and exosporium epitopes of Bacillus atrophaeus and Bacillus anthracis spores.« less

Recent progress in the development of anthrax vaccines.

PubMed

Kaur, Manpreet; Bhatnagar, Rakesh

2011-12-01

Bacillus anthracis is the etiological agent of anthrax. Although anthrax is primarily an epizootic disease; humans are at risk for contracting anthrax. The potential use of B. anthracis spores as biowarfare agent has led to immense attention. Prolonged vaccination schedule of current anthrax vaccine and variable protection conferred; often leading to failure of therapy. This highlights the need for alternative anthrax countermeasures. A number of approaches are being investigated to substitute or supplement the existing anthrax vaccines. These relied on expression of Protective antigen (PA), the key protective immunogen; in bacterial or plant systems; or utilization of attenuated strains of B. anthracis for immunization. Few studies have established potential of domain IV of PA for immunization. Other targets including the spore, capsule, S-layer and anthrax toxin components have been investigated for imparting protective immunity. It has been shown that co-immunization of PA with domain I of lethal factor that binds PA resulted in higher antibody responses. Of the epitope based vaccines, the loop neutralizing determinant, in particular; elicited robust neutralizing antibody response and conferred 97% protection upon challenge. DNA vaccination resulted in varying degree of protection and seems a promising approach. Additionally, the applicability of monoclonal and therapeutic antibodies in the treatment of anthrax has also been demonstrated. The recent progress in the direction of anthrax prophylaxis has been evaluated in this review.

Progress and novel strategies in vaccine development and treatment of anthrax.

PubMed

Chitlaru, Theodor; Altboum, Zeev; Reuveny, Shaul; Shafferman, Avigdor

2011-01-01

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis. © 2010 John Wiley & Sons A/S.

A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection.

PubMed

Hepler, Robert W; Kelly, Rosemarie; McNeely, Tessie B; Fan, Hongxia; Losada, Maria C; George, Hugh A; Woods, Andrea; Cope, Leslie D; Bansal, Alka; Cook, James C; Zang, Gina; Cohen, Steven L; Wei, Xiaorong; Keller, Paul M; Leffel, Elizabeth; Joyce, Joseph G; Pitt, Louise; Schultz, Loren D; Jansen, Kathrin U; Kurtz, Myra

2006-03-06

Infection by Bacillus anthracis is preventable by prophylactic vaccination with several naturally derived and recombinant vaccine preparations. Existing data suggests that protection is mediated by antibodies directed against the protective antigen (PA) component of the anthrax toxin complex. PA is an 83-kDa protein cleaved in vivo to yield a biologically active 63-kDa protein. In an effort to evaluate the potential of yeast as an expression system for the production of recombinant PA, and to determine if the yeast-purified rPA63 can protect from a lethal inhalational challenge, the sequence of the 63-kDa form of PA was codon-optimized and expressed in the yeast Saccharomyces cerevisiae. Highly purified rPA63 isolated from Saccharomyces under denaturing conditions demonstrated reduced biological activity in a macrophage-killing assay compared to non-denatured rPA83 purified from Escherichia coli. Rabbits and non-human primates (NHP) immunized with rPA63 and later challenged with a lethal dose of B. anthracis spores were generally protected from infection. These results indicate that epitopes present in the 63-kDa from of PA can protect rabbits and non-human primates from a lethal spore challenge, and further suggest that a fully functional rPA63 is not required in order to provide these epitopes.

Low-Level Detection of a Bacillus Anthracis Simulant using Love-Wave Biosensors on 36 Degree YX LiTaO3

DOE Office of Scientific and Technical Information (OSTI.GOV)

BRANCH,DARREN W.; BROZIK,SUSAN M.

Crucial to low-level detection of biowarfare agents in aqueous environments is the mass sensitivity optimization of Love-wave acoustic sensors. The present work is an experimental study of 36{sup o} YX cut LiTaO{sub 3} based Love-wave devices for detection of pathogenic spores in aqueous conditions. Given that the detection limit (DL) of Love-wave based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene. To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording magnitude and phase shift acrossmore » each sensor. Polyimide had the lowest mass detection limit with an estimated value of 1-2 ng/cm{sup 2}, as compared to polystyrene where DL = 2.0 ng/cm{sup 2}. Suitable chemistries were used to orient antibodies on the Love-wave sensor using adsorbed protein G. The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated. The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B. thuringiensis B8 spores. Bacillus Subtilis spores were used as a negative control to determine whether significant non-specific binding would occur. Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation. This work demonstrates that Love-wave devices can be used to detect B. anthracis simulant below reported infectious levels.« less

Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

PubMed

Heffron, Jared D; Orsburn, Benjamin; Popham, David L

2009-04-01

The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

The effect of deletion of the edema factor on Bacillus anthracis pathogenicity in guinea pigs and rabbits.

PubMed

Levy, Haim; Weiss, Shay; Altboum, Zeev; Schlomovitz, Josef; Rothschild, Nili; Glinert, Itai; Sittner, Assa; Kobiler, David

2012-01-01

Bacillus anthracis secretes three major components, which assemble into two bipartite toxins: lethal toxin (LT), composed of lethal factor (LF) and protective antigen (PA) and edema toxin (ET), composed of edema factor (EF) and PA. EF is a potent calmodulin-dependent adenylate cyclase, which is internalized into the target cell following PA binding. Once inside the cell, EF elevates cAMP levels, interrupting intracellular signaling. Effects of ET were demonstrated on monocytes, neutrophils and T-cells. In an earlier work we demonstrated that a deletion of LF in a fully virulent strain had no effect in guinea pigs and a significant, but not major, effect in the rabbit model. These results suggested that EF might play an important role in the development of infection and mortality following exposure to B. anthracis spores. To evaluate the role of EF in B. anthracis pathogenicity we deleted the cya gene, which encodes the EF protein, in the fully virulent Vollum strain. The Δcya mutant was fully virulent in the guinea pig model as determined by LD(50) experiments. In the rabbit model, when infected subcutaneously, the absence of EF had no effect on the virulence of the mutant. However an increase of two orders of magnitude in the LD(50) was demonstrated when the rabbits were infected by intranasal instillation accompanied with partial mortality and increased mean time to death. These results argue that in the guinea pig model the presence of one of the toxins, ET or LT is sufficient for the development of the infection. In the rabbit model ET plays a role in respiratory infection, most probably mediating the early steps of host colonization. Copyright © 2011 Elsevier Ltd. All rights reserved.

DESTRUCTION OF FRANCISELLA TULARENSIS AND YERSINIA PESTIS PERSISTENCE OF BACILLUS ANTHRACIS SPORES AND CLOSTRIDIUM BOTULINUM IN MUNICIPAL SOLID LANDFILL LEACHATES

EPA Science Inventory

The United States Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NHSRC) in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC) are evaluating the permanence of biological and chemi...

Monochloramine inactivation of bacterial select agents.

PubMed

Rose, Laura J; Rice, Eugene W; Hodges, Lisa; Peterson, Alicia; Arduino, Matthew J

2007-05-01

Seven species of bacterial select agents were tested for susceptibility to monochloramine. Under test conditions, the monochloramine routinely maintained in potable water would reduce six of the species by 2 orders of magnitude within 4.2 h. Bacillus anthracis spores would require up to 3.5 days for the same inactivation with monochloramine.

Transcriptional Profiling of Murine Organ Genes in Response to Infection with Bacillus anthracis Ames Spores

PubMed Central

Moen, Scott T.; Yeager, Linsey A.; Lawrence, William S.; Ponce, Cindy; Galindo, Cristi L.; Garner, Harold R.; Baze, Wallace B.; Suarez, Giovanni; Peterson, Johnny W.; Chopra, Ashok K.

2008-01-01

Bacillus anthracis is the gram positive, spore-forming etiological agent of anthrax, an affliction studied because of its importance as a potential bioweapon. Although in vitro transcriptional responses of macrophages to either spore or anthrax toxins have been previously reported, little is known regarding the impact of infection on gene expression in host tissues. We infected Swiss-Webster mice intranasally with 5 LD50 of B. anthracis virulent Ames spores and observed the global transcriptional profiles of various tissues over a 48 hr time period. RNA was extracted from spleen, lung, and heart tissues of infected and control mice and examined by Affymetrix GeneChip analysis. Approximately 580 host genes were significantly over or under expressed among the lung, spleen, and heart tissues at 8 hr and 48 hr time points. Expression of genes encoding for surfactant and major histocompatibility complex (MHC) presentation was diminished during the early phase of infection in lungs. By 48 hr, a significant number of genes were modulated in the heart, including up-regulation of calcium-binding related gene expression, and down-regulation of multiple genes related to cell adhesion, formation of the extracellular matrix, and the cell cytoskeleton. Interestingly, the spleen 8 hr post-infection showed striking increases in the expression of genes that encode hydrolytic enzymes, and these levels remained elevated throughout infection. Further, genes involving antigen presentation and interferon responses were down-regulated in the spleen at 8 hr. In late stages of infection, splenic genes related to the inflammatory response were up-regulated. This study is the first to describe the in vivo global transcriptional response of multiple organs during inhalational anthrax. Although numerous genes related to the host immunological response and certain protection mechanisms were up-regulated in these organs, a vast list of genes important for fully developing and maintaining this response were decreased. Additionally, the lung, spleen, and heart showed differential responses to the infection, further validating the demand for a better understanding of anthrax pathogenesis in order to design therapies against novel targets. PMID:18037264

Immunogenicity and efficacy of an anthrax/plague DNA fusion vaccine in a mouse model.

PubMed

Albrecht, Mark T; Eyles, Jim E; Baillie, Les W; Keane-Myers, Andrea M

2012-08-01

The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y . pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B. anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y. pestis. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Modeling the geographic distribution of Bacillus anthracis, the causative agent of anthrax disease, for the contiguous United States using predictive ecological [corrected] niche modeling.

PubMed

Blackburn, Jason K; McNyset, Kristina M; Curtis, Andrew; Hugh-Jones, Martin E

2007-12-01

The ecology and distribution of Bacillus anthracis is poorly understood despite continued anthrax outbreaks in wildlife and livestock throughout the United States. Little work is available to define the potential environments that may lead to prolonged spore survival and subsequent outbreaks. This study used the genetic algorithm for rule-set prediction modeling system to model the ecological niche for B. anthracis in the contiguous United States using wildlife and livestock outbreaks and several environmental variables. The modeled niche is defined by a narrow range of normalized difference vegetation index, precipitation, and elevation, with the geographic distribution heavily concentrated in a narrow corridor from southwest Texas northward into the Dakotas and Minnesota. Because disease control programs rely on vaccination and carcass disposal, and vaccination in wildlife remains untenable, understanding the distribution of B. anthracis plays an important role in efforts to prevent/eradicate the disease. Likewise, these results potentially aid in differentiating endemic/natural outbreaks from industrial-contamination related outbreaks or bioterrorist attacks.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-06-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

PubMed Central

Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

1997-01-01

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines. PMID:9169728

Epidemiologic Responses to Anthrax Outbreaks: A Review of Field Investigations, 1950–2001

PubMed Central

Bales, Michael E.; Brachman, Philip S.; Kaufmann, Arnold F.; Klatsky, Peter C.; Ashford, David A.

2002-01-01

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. PMID:12396934

A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production

PubMed Central

Dale, Jennifer L.; Raynor, Malik J.; Ty, Maureen C.; Hadjifrangiskou, Maria; Koehler, Theresa M.

2018-01-01

Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075, an atxA-regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 “skiA” for “sporulation kinase inhibitor.” Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development. PMID:29599764

A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production.

PubMed

Dale, Jennifer L; Raynor, Malik J; Ty, Maureen C; Hadjifrangiskou, Maria; Koehler, Theresa M

2018-01-01

Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075 , an atxA -regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 " skiA " for "sporulation kinase inhibitor." Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.

Decontamination Efficacy of Three Commercial-Off-The-Shelf (COTS) Sporicidal Disinfectants on Medium-Sized Panels Contaminated with Surrogate Spores of Bacillus anthracis

PubMed Central

Sabol, Jonathan P.

2014-01-01

In the event of a wide area release and contamination of a biological agent in an outdoor environment and to building exteriors, decontamination is likely to consume the Nation’s remediation capacity, requiring years to cleanup, and leading to incalculable economic losses. This is in part due to scant body of efficacy data on surface areas larger than those studied in a typical laboratory (5×10-cm), resulting in low confidence for operational considerations in sampling and quantitative measurements of prospective technologies recruited in effective cleanup and restoration response. In addition to well-documented fumigation-based cleanup efforts, agencies responsible for mitigation of contaminated sites are exploring alternative methods for decontamination including combinations of disposal of contaminated items, source reduction by vacuuming, mechanical scrubbing, and low-technology alternatives such as pH-adjusted bleach pressure wash. If proven effective, a pressure wash-based removal of Bacillus anthracis spores from building surfaces with readily available equipment will significantly increase the readiness of Federal agencies to meet the daunting challenge of restoration and cleanup effort following a wide-area biological release. In this inter-agency study, the efficacy of commercial-of-the-shelf sporicidal disinfectants applied using backpack sprayers was evaluated in decontamination of spores on the surfaces of medium-sized (∼1.2 m2) panels of steel, pressure-treated (PT) lumber, and brick veneer. Of the three disinfectants, pH-amended bleach, Peridox, and CASCAD evaluated; CASCAD was found to be the most effective in decontamination of spores from all three panel surface types. PMID:24940605

Determination of serum IgG antibodies to Bacillus anthracis protective antigen in environmental sampling workers using a fluorescent covalent microsphere immunoassay.

PubMed

Biagini, R E; Sammons, D L; Smith, J P; Page, E H; Snawder, J E; Striley, C A F; MacKenzie, B A

2004-08-01

To evaluate potential exposure to Bacillis anthracis (Ba) spores in sampling/decontamination workers in the aftermath of an anthrax terror attack. Fifty six serum samples were obtained from workers involved in environmental sampling for Ba spores at the American Media, Inc. (AMI) building in Boca Raton, FL after the anthrax attack there in October 2001. Nineteen sera were drawn from individuals both pre-entry and several weeks after entrance into the building. Nine sera each were drawn from unique individuals at the pre-entry and follow up blood draws. Thirteen donor control sera were also evaluated. Individuals were surveyed for Ba exposure by measurement of serum Ba anti-protective antigen (PA) specific IgG antibodies using a newly developed fluorescent covalent microsphere immunoassay (FCMIA). Four sera gave positive anti-PA IgG results (defined as anti-PA IgG concentrations > or = the mean microg/ml anti-PA IgG from donor control sera (n = 13 plus 2 SD which were also inhibited > or = 85% when the serum was pre-adsorbed with PA). The positive sera were the pre-entry and follow up samples of two workers who had received their last dose of anthrax vaccine in 2000. It appears that the sampling/decontamination workers of the present study either had insufficient exposure to Ba spores to cause the production of anti-PA IgG antibodies or they were exposed to anthrax spores without producing antibody. The FCMIA appears to be a fast, sensitive, accurate, and precise method for the measurement of anti-PA IgG antibodies.

Anthrax toxin.

PubMed

Bhatnagar, R; Batra, S

2001-01-01

Anthrax is primarily a disease of herbivores caused by gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63 kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis.

Decontamination of Anthrax spores in critical infrastructure and critical assets.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boucher, Raymond M.; Crown, Kevin K.; Tucker, Mark David

2010-05-01

Decontamination of anthrax spores in critical infrastructure (e.g., subway systems, major airports) and critical assets (e.g., the interior of aircraft) can be challenging because effective decontaminants can damage materials. Current decontamination methods require the use of highly toxic and/or highly corrosive chemical solutions because bacterial spores are very difficult to kill. Bacterial spores such as Bacillus anthracis, the infectious agent of anthrax, are one of the most resistant forms of life and are several orders of magnitude more difficult to kill than their associated vegetative cells. Remediation of facilities and other spaces (e.g., subways, airports, and the interior of aircraft)more » contaminated with anthrax spores currently requires highly toxic and corrosive chemicals such as chlorine dioxide gas, vapor- phase hydrogen peroxide, or high-strength bleach, typically requiring complex deployment methods. We have developed a non-toxic, non-corrosive decontamination method to kill highly resistant bacterial spores in critical infrastructure and critical assets. A chemical solution that triggers the germination process in bacterial spores and causes those spores to rapidly and completely change to much less-resistant vegetative cells that can be easily killed. Vegetative cells are then exposed to mild chemicals (e.g., low concentrations of hydrogen peroxide, quaternary ammonium compounds, alcohols, aldehydes, etc.) or natural elements (e.g., heat, humidity, ultraviolet light, etc.) for complete and rapid kill. Our process employs a novel germination solution consisting of low-cost, non-toxic and non-corrosive chemicals. We are testing both direct surface application and aerosol delivery of the solutions. A key Homeland Security need is to develop the capability to rapidly recover from an attack utilizing biological warfare agents. This project will provide the capability to rapidly and safely decontaminate critical facilities and assets to return them to normal operations as quickly as possible, sparing significant economic damage by re-opening critical facilities more rapidly and safely. Facilities and assets contaminated with Bacillus anthracis (i.e., anthrax) spores can be decontaminated with mild chemicals as compared to the harsh chemicals currently needed. Both the 'germination' solution and the 'kill' solution are constructed of 'off-the-shelf,' inexpensive chemicals. The method can be utilized by directly spraying the solutions onto exposed surfaces or by application of the solutions as aerosols (i.e., small droplets), which can also reach hidden surfaces.« less

Bio-Response Operational Testing and Evaluation (BOTE) ...

EPA Pesticide Factsheets

Report and technical brief and informational video The Bio-response Operational Testing and Evaluation (BOTE) Project was a multi-agency effort designed to operationally test and evaluate, at the scale of a moderately sized building, a response to a B. anthracis spore release from initial public health and law enforcement investigation through environmental remediation.

Decontamination Efficacy of Three Commercial Off-the-Shelf Sporicidal Agents on Medium-Sized Panels Contaminated with Surrogates of Bacillus anthracis

DTIC Science & Technology

2012-04-01

chlorine dioxide (CD) or vapor hydrogen peroxide ( VHP ). A wide-area release and contamination of building exteriors and the outdoors would likely...from the panels. Depending on the surface composition and the decontamination technology tested, viable spore recovery from the panels varied after

Monochloramine Inactivation of Bacterial Select Agents▿

PubMed Central

Rose, Laura J.; Rice, Eugene W.; Hodges, Lisa; Peterson, Alicia; Arduino, Matthew J.

2007-01-01

Seven species of bacterial select agents were tested for susceptibility to monochloramine. Under test conditions, the monochloramine routinely maintained in potable water would reduce six of the species by 2 orders of magnitude within 4.2 h. Bacillus anthracis spores would require up to 3.5 days for the same inactivation with monochloramine. PMID:17400782

Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.

PubMed

Bensman, M D; Mackie, R S; Minter, Z A; Gutting, B W

2012-08-01

 The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species.  Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria.  The data suggested sera can have a significant impact on germination and growth of Sterne bacteria.  These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

A Novel Immunogenic Spore Coat-Associated Protein in Bacillus Anthracis: Characterization via Proteomics Approaches and a Vector-Based Vaccine System

PubMed Central

Liu, Yu-Tsueng; Lin, Shwu-Bin; Huang, Cheng-Po; Huang, Chun-Ming

2007-01-01

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An E. coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines. PMID:18029197

Role of mechanical vs. chemical action in the removal of adherent Bacillus spores during CIP procedures.

PubMed

Faille, C; Bénézech, T; Blel, W; Ronse, A; Ronse, G; Clarisse, M; Slomianny, C

2013-04-01

This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60°C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry. Copyright © 2012 Elsevier Ltd. All rights reserved.

Forensic Application of Microbiological Culture Analysis To Identify Mail Intentionally Contaminated with Bacillus anthracis Spores†

PubMed Central

Beecher, Douglas J.

2006-01-01

The discovery of a letter intentionally filled with dried Bacillus anthracis spores in the office of a United States senator prompted the collection and quarantine of all mail in congressional buildings. This mail was subsequently searched for additional intentionally contaminated letters. A microbiological sampling strategy was used to locate heavy contamination within the 642 separate plastic bags containing the mail. Swab sampling identified 20 bags for manual and visual examination. Air sampling within the 20 bags indicated that one bag was orders of magnitude more contaminated than all the others. This bag contained a letter addressed to Senator Patrick Leahy that had been loaded with dried B. anthracis spores. Microbiological sampling of compartmentalized batches of mail proved to be efficient and relatively safe. Efficiency was increased by inoculating culture media in the hot zone rather than transferring swab samples to a laboratory for inoculation. All mail sampling was complete within 4 days with minimal contamination of the sampling environment or personnel. However, physically handling the intentionally contaminated letter proved to be exceptionally hazardous, as did sorting of cross-contaminated mail, which resulted in generation of hazardous aerosol and extensive contamination of protective clothing. Nearly 8 × 106 CFU was removed from the most highly cross-contaminated piece of mail found. Tracking data indicated that this and other heavily contaminated envelopes had been processed through the same mail sorting equipment as, and within 1 s of, two intentionally contaminated letters. PMID:16885280

Efficacy and immunogenicity of single-dose AdVAV intranasal anthrax vaccine compared to anthrax vaccine absorbed in an aerosolized spore rabbit challenge model.

PubMed

Krishnan, Vyjayanthi; Andersen, Bo H; Shoemaker, Christine; Sivko, Gloria S; Tordoff, Kevin P; Stark, Gregory V; Zhang, Jianfeng; Feng, Tsungwei; Duchars, Matthew; Roberts, M Scot

2015-04-01

AdVAV is a replication-deficient adenovirus type 5-vectored vaccine expressing the 83-kDa protective antigen (PA83) from Bacillus anthracis that is being developed for the prevention of disease caused by inhalation of aerosolized B. anthracis spores. A noninferiority study comparing the efficacy of AdVAV to the currently licensed Anthrax Vaccine Absorbed (AVA; BioThrax) was performed in New Zealand White rabbits using postchallenge survival as the study endpoint (20% noninferiority margin for survival). Three groups of 32 rabbits were vaccinated with a single intranasal dose of AdVAV (7.5 × 10(7), 1.5 × 10(9), or 3.5 × 10(10) viral particles). Three additional groups of 32 animals received two doses of either intranasal AdVAV (3.5 × 10(10) viral particles) or intramuscular AVA (diluted 1:16 or 1:64) 28 days apart. The placebo group of 16 rabbits received a single intranasal dose of AdVAV formulation buffer. All animals were challenged via the inhalation route with a targeted dose of 200 times the 50% lethal dose (LD50) of aerosolized B. anthracis Ames spores 70 days after the initial vaccination and were followed for 3 weeks. PA83 immunogenicity was evaluated by validated toxin neutralizing antibody and serum anti-PA83 IgG enzyme-linked immunosorbent assays (ELISAs). All animals in the placebo cohort died from the challenge. Three of the four AdVAV dose cohorts tested, including two single-dose cohorts, achieved statistical noninferiority relative to the AVA comparator group, with survival rates between 97% and 100%. Vaccination with AdVAV also produced antibody titers with earlier onset and greater persistence than vaccination with AVA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of early immune response-survival relationship in cynomolgus macaques after Anthrax Vaccine Adsorbed vaccination and Bacillus anthracis spore challenge.

PubMed

Sivko, G S; Stark, G V; Tordoff, K P; Taylor, K L; Glaze, E; VanRaden, M; Schiffer, J M; Hewitt, J A; Quinn, C P; Nuzum, E O

2016-12-12

Anthrax Vaccine Adsorbed (AVA, BioThrax) is approved by the US Food and Drug Administration for post-exposure prophylaxis (PEP) of anthrax in adults. The PEP schedule is 3 subcutaneous (SC) doses (0, 14 and 28 days), in conjunction with a 60 day course of antimicrobials. The objectives of this study were to understand the onset of protection from AVA PEP vaccination and to assess the potential for shortening the duration of antimicrobial treatment (http://www.phe.gov/Preparedness/mcm/phemce/Documents/2014-phemce-sip.pdf). We determined the efficacy against inhalation anthrax in nonhuman primates (NHP) of the first two doses of the PEP schedule by infectious challenge at the time scheduled for receipt of the third PEP dose (Day 28). Forty-eight cynomolgus macaques were randomized to five groups and vaccinated with serial dilutions of AVA on Days 0 and 14. NHP were exposed to Bacillus anthracis Ames spores on Day 28 (target dose 200 LD 50 equivalents). Anti-protective antigen (PA) IgG and toxin neutralizing antibody (TNA) responses to vaccination and in post-challenge survivors were determined. Post-challenge blood and selected tissue samples were assessed for B. anthracis at necropsy or end of study (Day 56). Pre-challenge humoral immune responses correlated with survival, which ranged from 24 to 100% survival depending on vaccination group. Surviving, vaccinated animals had elevated anti-PA IgG and TNA levels for the duration of the study, were abacteremic, exhibited no apparent signs of infection, and had no gross or microscopic lesions. However, survivors had residual spores in lung tissues. We conclude that the first two doses of the PEP schedule provide high levels of protection by the scheduled timing of the third dose. These data may also support consideration of a shorter duration PEP antimicrobial regimen. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using Telemetry Data to Refine Endpoints for New Zealand White Rabbits Challenged with Bacillus anthracis.

PubMed

Dawson, David G; Bower, Kristin A; Burnette, Candace N; Holt, Rebecca K; Swearengen, James R; Dabisch, Paul A; Scorpio, Angelo

2017-11-01

We used a continuous-monitoring digital telemetry system to investigate temperature response in New Zealand White rabbits after inhalation or subcutaneous challenge with Bacillus anthracis. Two spore preparations of B. anthracis Ames A2084 were evaluated by using a nose-only inhalation model, and 2 strains, B. anthracis Ames A2084 and B. anthracis UT500, were evaluated in a subcutaneous model. Animal body temperature greater than 3 SD above the mean baseline temperature was considered a significant increase in body temperature (SIBT). All rabbits that exhibited SIBT after challenge by either route of infection or bacterial strain eventually died or were euthanized due to infection, and all rabbits that died or were euthanized due to infection exhibited SIBT during the course of disease. The time at onset of SIBT preceded clinical signs of disease in 94% of the rabbits tested by as long as 2 days. In addition, continuous temperature monitoring facilitated discrimination between the 2 B. anthracis strains with regard to the time interval between SIBT and death. These data suggest that for the New Zealand White rabbit anthrax model, SIBT is a reliable indicator of infection, is predictive of experimental outcome in the absence of treatment, and is measurable prior to the appearance of more severe signs of disease. The use of digital telemetry to monitor infectious disease course in animal models of anthrax can potentially be used in conjunction with other clinical score metrics to refine endpoint euthanasia criteria.

Processing Protocol for Soil Samples Potentially ...

EPA Pesticide Factsheets

Method Operating Procedures This protocol describes the processing steps for 45 g and 9 g soil samples potentially contaminated with Bacillus anthracis spores. The protocol is designed to separate and concentrate the spores from bulk soil down to a pellet that can be used for further analysis. Soil extraction solution and mechanical shaking are used to disrupt soil particle aggregates and to aid in the separation of spores from soil particles. Soil samples are washed twice with soil extraction solution to maximize recovery. Differential centrifugation is used to separate spores from the majority of the soil material. The 45 g protocol has been demonstrated by two laboratories using both loamy and sandy soil types. There were no significant differences overall between the two laboratories for either soil type, suggesting that the processing protocol would be robust enough to use at multiple laboratories while achieving comparable recoveries. The 45 g protocol has demonstrated a matrix limit of detection at 14 spores/gram of soil for loamy and sandy soils.

Fighting Ebola with novel spore decontamination technologies for the military.

PubMed

Doona, Christopher J; Feeherry, Florence E; Kustin, Kenneth; Olinger, Gene G; Setlow, Peter; Malkin, Alexander J; Leighton, Terrance

2015-01-01

Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC's novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2.

Fighting Ebola with novel spore decontamination technologies for the military

PubMed Central

Doona, Christopher J.; Feeherry, Florence E.; Kustin, Kenneth; Olinger, Gene G.; Setlow, Peter; Malkin, Alexander J.; Leighton, Terrance

2015-01-01

Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC’s novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2. PMID:26322021

Decon Green (trademark)

DTIC Science & Technology

2004-11-17

Bacillus stearothermophilus spores, a species considered extremely resistant to peroxide sterilants . As seen for Decon GreenTM Classic, New Decon...Additional data is given for Bacillus anthracis and Bacillus stearothermophilus demonstrating that Decon GreenTM is also effective against bio...GreenTM retains excellent bio decon efficacy. TABLE 4. Decontamination of Bacillus stearothermophilus by New Decon GreenTM Challenge CFU Recovered

Structure of the Anthrax Research Literature

DTIC Science & Technology

2006-01-01

4; identification 4; staphylococcus - aureus 3; pharmacology & pharmacy 3; microbiology 3; pharmacology & pharmacy 2; biotechnology & applied...proteins 4; microbiology 4; escherichia-coli 4; bacillus-anthracis 4; cell-wall 3; staphylococcus - aureus 3 Country usa 9; france 7; england 2...pathogen detection using a microchip PCR array Instrument 199 In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence

Bacillus Anthracis Spore Interactions with Mammalian Cells: Relationship Between Germination State and the Outcome of in Vitro Infections

DTIC Science & Technology

2011-02-28

unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Report Documentation Page Form ApprovedOMB... Peacock S, Belton FC: Observations on the prophylaxis of experimental pulmonary anthrax in the monkey. J Hyg (Lond) 1956, 54(1):28-36. 8. Cleret A

Reaerosolization of Fluidized Spores in Ventilation Systems▿

PubMed Central

Krauter, Paula; Biermann, Arthur

2007-01-01

This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of Bacillus atrophaeus, a nonpathogenic surrogate for Bacillus anthracis, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 1010 to 1011 viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10−3 and 10−5 per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours. PMID:17293522

Encapsulated Bacillus anthracis interacts closely with liver endothelium.

PubMed

Piris-Gimenez, Alejandro; Corre, Jean-Philippe; Jouvion, Gregory; Candela, Thomas; Khun, Huot; Goossens, Pierre L

2009-11-01

The Bacillus anthracis poly-gamma-D-glutamate capsule is essential for virulence. It impedes phagocytosis and protects bacilli from the immune system, thus promoting systemic dissemination. To further define the virulence mechanisms brought into play by the capsule, we characterized the interactions between encapsulated nontoxinogenic B. anthracis and its host in vivo through histological analysis, perfusion, and competition experiments with purified capsule. Clearance of encapsulated bacilli from the blood was rapid (>90% clearance within 5 min), with 75% of the bacteria being trapped in the liver. Competition experiments with purified capsule polyglutamate inhibited this interaction. At the septicemic phase of cutaneous infection with spores, the encapsulated bacilli were trapped in the vascular spaces of the liver and interacted closely with the liver endothelium in the sinusoids and terminal and portal veins. They often grow as microcolonies containing capsular material shed by the bacteria. We show that, in addition to its inhibitory effect on the interaction with the immune system, the capsule surrounding B. anthracis plays an active role in mediating the trapping of the bacteria within the liver and may thus contribute to anthrax pathogenesis. Because other microorganisms produce polyglutamate, it may also represent a general mechanism of virulence or in vivo survival.

Alternative pre-approved and novel therapies for the treatment of anthrax.

PubMed

Head, Breanne M; Rubinstein, Ethan; Meyers, Adrienne F A

2016-11-03

Bacillus anthracis, the causative agent of anthrax, is a spore forming and toxin producing rod-shaped bacterium that is classified as a category A bioterror agent. This pathogenic microbe can be transmitted to both animals and humans. Clinical presentation depends on the route of entry (direct contact, ingestion, injection or aerosolization) with symptoms ranging from isolated skin infections to more severe manifestations such as cardiac or pulmonary shock, meningitis, and death. To date, anthrax is treatable if antibiotics are administered promptly and continued for 60 days. However, if treatment is delayed or administered improperly, the patient's chances of survival are decreased drastically. In addition, antibiotics are ineffective against the harmful anthrax toxins and spores. Therefore, alternative therapeutics are essential. In this review article, we explore and discuss advances that have been made in anthrax therapy with a primary focus on alternative pre-approved and novel antibiotics as well as anti-toxin therapies. A literature search was conducted using the University of Manitoba search engine. Using this search engine allowed access to a greater variety of journals/articles that would have otherwise been restricted for general use. In order to be considered for discussion for this review, all articles must have been published later than 2009. The alternative pre-approved antibiotics demonstrated high efficacy against B. anthracis both in vitro and in vivo. In addition, the safety profile and clinical pharmacology of these drugs were already known. Compounds that targeted underexploited bacterial processes (DNA replication, RNA synthesis, and cell division) were also very effective in combatting B. anthracis. In addition, these novel compounds prevented bacterial resistance. Targeting B. anthracis virulence, more specifically the anthrax toxins, increased the length of which treatment could be administered. Several novel and pre-existing antibiotics, as well as toxin inhibitors, have shown increasing promise. A combination treatment that targets both bacterial growth and toxin production would be ideal and probably necessary for effectively combatting this armed bacterium.

Bioterrorism-related Inhalational Anthrax in an Elderly Woman, Connecticut, 2001

PubMed Central

Mead, Paul; Armstrong, Gregory L.; Painter, John; Kelley, Katherine A.; Hoffmaster, Alex R.; Mayo, Donald; Barden, Diane; Ridzon, Renee; Parashar, Umesh; Teshale, Eyasu Habtu; Williams, Jen; Noviello, Stephanie; Perz, Joseph F.; Mast, Eric E.; Swerdlow, David L.; Hadler, James L.

2003-01-01

On November 20, 2001, inhalational anthrax was confirmed in an elderly woman from rural Connecticut. To determine her exposure source, we conducted an extensive epidemiologic, environmental, and laboratory investigation. Molecular subtyping showed that her isolate was indistinguishable from isolates associated with intentionally contaminated letters. No samples from her home or community yielded Bacillus anthracis, and she received no first-class letters from facilities known to have processed intentionally contaminated letters. Environmental sampling in the regional Connecticut postal facility yielded B. anthracis spores from 4 (31%) of 13 sorting machines. One extensively contaminated machine primarily processes bulk mail. A second machine that does final sorting of bulk mail for her zip code yielded B. anthracis on the column of bins for her carrier route. The evidence suggests she was exposed through a cross-contaminated bulk mail letter. Such cross-contamination of letters and postal facilities has implications for managing the response to future B. anthracis–contaminated mailings. PMID:12781007

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

PubMed

Parthasarathy, N; Saksena, R; Kováč, P; Deshazer, D; Peacock, S J; Wuthiekanun, V; Heine, H S; Friedlander, A M; Cote, C K; Welkos, S L; Adamovicz, J J; Bavari, S; Waag, D M

2008-11-03

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Summary Document: Restoration Plan for Major Airports after a Bioterrorist Attack

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raber, E

2007-01-11

This document provides general guidelines for developing a Restoration Plan for a major airport following release of a biological warfare agent. San Francisco International Airport was selected as the example airport during development of the Plan to illustrate specific details. The spore forming bacterium Bacillus anthracis was selected as the biological agent of primary concern because it is the most difficult of known bioterrorism agents to inactivate and is considered to be one of the agents most likely to be used as a biological weapon. The focus of the Plan is on activities associated with the Characterization, Remediation, and Clearancemore » Phases that are defined herein. Activities associated with the Notification and First-Response Phases are briefly discussed in Appendixes A and B, respectively. In addition to the main text of this Plan and associated appendixes, a data supplement was developed specifically for San Francisco International Airport. Requests for the data supplement must be made directly to the Emergency Planning Operations Division of San Francisco International Airport.« less

Obiltoxaximab Prevents Disseminated Bacillus anthracis Infection and Improves Survival during Pre- and Postexposure Prophylaxis in Animal Models of Inhalational Anthrax.

PubMed

Yamamoto, Brent J; Shadiack, Annette M; Carpenter, Sarah; Sanford, Daniel; Henning, Lisa N; Gonzales, Nestor; O'Connor, Edward; Casey, Leslie S; Serbina, Natalya V

2016-10-01

The Centers for Disease Control and Prevention recommend adjunctive antitoxins when systemic anthrax is suspected. Obiltoxaximab, a monoclonal antibody against protective antigen (PA), is approved for treatment of inhalational anthrax in combination with antibiotics and for prophylaxis when alternative therapies are not available. The impact of toxin neutralization with obiltoxaximab during pre- and postexposure prophylaxis was explored, and efficacy results that supported the prophylaxis indication are presented here. New Zealand White rabbits and cynomolgus macaques received obiltoxaximab as a single intramuscular or intravenous dose of 2 to 16 mg/kg of body weight at various times relative to Bacillus anthracis aerosol spore challenge. The primary endpoint was survival, and effect of treatment timing was explored. In rabbits, obiltoxaximab administration 9 h postchallenge singly or combined with a 5-day levofloxacin regimen protected 89% to 100% of animals compared to 33% with levofloxacin monotherapy. In cynomolgus macaques, a single intramuscular dose of 16 mg/kg obiltoxaximab led to 100% survival when given 1 to 3 days preexposure and 83% to 100% survival when given 18 to 24 h postexposure and prior to systemic bacteremia onset. Obiltoxaximab administration after bacteremia onset resulted in lower (25% to 50%) survival rates reflective of treatment setting. Prophylactic administration of obiltoxaximab before spore challenge or to spore-challenged animals before systemic bacterial dissemination is efficacious in promoting survival, ameliorating toxemia, and inhibiting bacterial spread to the periphery. Copyright © 2016 Yamamoto et al.

Obiltoxaximab Prevents Disseminated Bacillus anthracis Infection and Improves Survival during Pre- and Postexposure Prophylaxis in Animal Models of Inhalational Anthrax

PubMed Central

Yamamoto, Brent J.; Shadiack, Annette M.; Carpenter, Sarah; Sanford, Daniel; Henning, Lisa N.; Gonzales, Nestor; O'Connor, Edward; Casey, Leslie S.

2016-01-01

The Centers for Disease Control and Prevention recommend adjunctive antitoxins when systemic anthrax is suspected. Obiltoxaximab, a monoclonal antibody against protective antigen (PA), is approved for treatment of inhalational anthrax in combination with antibiotics and for prophylaxis when alternative therapies are not available. The impact of toxin neutralization with obiltoxaximab during pre- and postexposure prophylaxis was explored, and efficacy results that supported the prophylaxis indication are presented here. New Zealand White rabbits and cynomolgus macaques received obiltoxaximab as a single intramuscular or intravenous dose of 2 to 16 mg/kg of body weight at various times relative to Bacillus anthracis aerosol spore challenge. The primary endpoint was survival, and effect of treatment timing was explored. In rabbits, obiltoxaximab administration 9 h postchallenge singly or combined with a 5-day levofloxacin regimen protected 89% to 100% of animals compared to 33% with levofloxacin monotherapy. In cynomolgus macaques, a single intramuscular dose of 16 mg/kg obiltoxaximab led to 100% survival when given 1 to 3 days preexposure and 83% to 100% survival when given 18 to 24 h postexposure and prior to systemic bacteremia onset. Obiltoxaximab administration after bacteremia onset resulted in lower (25% to 50%) survival rates reflective of treatment setting. Prophylactic administration of obiltoxaximab before spore challenge or to spore-challenged animals before systemic bacterial dissemination is efficacious in promoting survival, ameliorating toxemia, and inhibiting bacterial spread to the periphery. PMID:27431219

Evaluation of PCR Systems for Field Screening of Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.

There is little published data on the performance of hand-portable polymerase chain reaction (PCR) instruments that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated five commercially available hand-portable PCR instruments for detection of Bacillus anthracis (Ba). We designed a cost-effective, statistically-based test plan that allows instruments to be evaluated at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) on the probability of detection (POD) at confidence levels of 80-95%. We assessed specificity using purified genomic DNA from 13 Ba strains and 18 Bacillus near neighbors, interference with 22more » common hoax powders encountered in the field, and PCR inhibition when Ba spores were spiked into these powders. Our results indicated that three of the five instruments achieved >0.95 LCB on the POD with 95% confidence at test concentrations of 2,000 genome equivalents/mL (comparable to 2,000 spores/mL), displaying more than sufficient sensitivity for screening suspicious powders. These instruments exhibited no false positive results or PCR inhibition with common hoax powders, and reliably detected Ba spores spiked into common hoax powders, though some issues with instrument controls were observed. Our testing approach enables efficient instrument performance testing to a statistically rigorous and cost-effective test plan to generate performance data that will allow users to make informed decisions regarding the purchase and use of biodetection equipment in the field.« less

Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States.

PubMed Central

Jernigan, J. A.; Stephens, D. S.; Ashford, D. A.; Omenaca, C.; Topiel, M. S.; Galbraith, M.; Tapper, M.; Fisk, T. L.; Zaki, S.; Popovic, T.; Meyer, R. F.; Quinn, C. P.; Harper, S. A.; Fridkin, S. K.; Sejvar, J. J.; Shepard, C. W.; McConnell, M.; Guarner, J.; Shieh, W. J.; Malecki, J. M.; Gerberding, J. L.; Hughes, J. M.; Perkins, B. A.

2001-01-01

From October 4 to November 2, 2001, the first 10 confirmed cases of inhalational anthrax caused by intentional release of Bacillus anthracis were identified in the United States. Epidemiologic investigation indicated that the outbreak, in the District of Columbia, Florida, New Jersey, and New York, resulted from intentional delivery of B. anthracis spores through mailed letters or packages. We describe the clinical presentation and course of these cases of bioterrorism-related inhalational anthrax. The median age of patients was 56 years (range 43 to 73 years), 70% were male, and except for one, all were known or believed to have processed, handled, or received letters containing B. anthracis spores. The median incubation period from the time of exposure to onset of symptoms, when known (n=6), was 4 days (range 4 to 6 days). Symptoms at initial presentation included fever or chills (n=10), sweats (n=7), fatigue or malaise (n=10), minimal or nonproductive cough (n=9), dyspnea (n=8), and nausea or vomiting (n=9). The median white blood cell count was 9.8 X 10(3)/mm(3) (range 7.5 to 13.3), often with increased neutrophils and band forms. Nine patients had elevated serum transaminase levels, and six were hypoxic. All 10 patients had abnormal chest X-rays; abnormalities included infiltrates (n=7), pleural effusion (n=8), and mediastinal widening (seven patients). Computed tomography of the chest was performed on eight patients, and mediastinal lymphadenopathy was present in seven. With multidrug antibiotic regimens and supportive care, survival of patients (60%) was markedly higher (<15%) than previously reported. PMID:11747719

Advances in the development of next-generation anthrax vaccines.

PubMed

Friedlander, Arthur M; Little, Stephen F

2009-11-05

Anthrax, a disease of herbivores, only rarely infects humans. However, the threat of using Bacillus anthracis, the causative agent, to intentionally produce disease has been the impetus for development of next-generation vaccines. Two licensed vaccines have been available for human use for several decades. These are composed of acellular culture supernatants containing the protective antigen (PA) component of the anthrax toxins. In this review we summarize the various approaches used to develop improved vaccines. These efforts have included the use of PA with newer adjuvants and delivery systems, including bacterial and viral vectors and DNA vaccines. Attempts to broaden the protection afforded by PA-based vaccines have focused on adding other B. anthracis components, including spore and capsule antigens.

Treatment of Anthrax Disease Frequently Asked Questions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judd, Kathleen S.; Young, Joan E.; Lesperance, Ann M.

2010-05-14

This document provides a summary of Frequently Asked Questions (FAQs) on the treatment of anthrax disease caused by a wide-area release of Bacillus anthracis spores as an act bioterrorism. These FAQs are intended to provide the public health and medical community, as well as others, with guidance and communications to support the response and long-term recovery from an anthrax event.

First detection of Bacillus anthracis in feces of free-ranging raptors from central Argentina.

PubMed

Saggese, Miguel D; Noseda, Ramón P; Uhart, Marcela M; Deem, Sharon L; Ferreyra, Hebe; Romano, Marcelo C; Ferreyra-Armas, María C; Hugh-Jones, Martin

2007-01-01

Prevalence of anthrax spores in feces of raptors was determined from samples collected in November-December 2000 and April-May 2001 in an agricultural region of Santa Fé province, Argentina. Feces were tested from 48 birds of six raptor species. One of 14 chimango caracaras (Milvago chimango) and one of eight road-side hawks (Buteo magnirostris) tested positive. The prevalence of Bacillus anthracis spores in feces for the six species was 4% (n=48). The prevalence was 7% (n=14) for chimango caracaras, 13% for road-side hawks (n=8), and 0% for the remaining species (Burrowing owl [Speotyto cunicularia] [n=17], Swainson's hawk [Buteo swainsoni] [n=3], Aplomado falcon [Falco femoralis] [n=2], and American kestrel [Falco sparverius] [n=4]). Grouped by their feeding habits, prevalence for scavenger species was not significantly different than for predators (7% vs. 3%, P>0.999). This study provides evidence that in central Argentina scavenger and non-scavenger raptors may have a role in the epidemiology of anthrax. Long-term studies to determine the extent of this potential involvement in the epidemiology of anthrax in central Argentina are required.

Design and characterization of a magnetoelastic sensor for the detection of biological agents

NASA Astrophysics Data System (ADS)

Shen, Wen; Mathison, Leslie C.; Petrenko, Valery A.; Chin, Bryan A.

2010-01-01

This paper presents the design and development of a free-standing, magnetoelastic biosensor. The detection principle is presented and various resonance characteristics of the sensor are discussed. Experimental measurements of the sensor resonance frequencies agree with theoretical predictions. The influence of the external magnetic field on the resonance behaviour of the sensor was studied and the optimum dc magnetic fields for best sensitivity in air and in water solutions for 2000 × 400 × 15 µm (2 mm) sensors and 1000 × 200 × 15 µm (1 mm) size sensors were determined to be 75 Oe and 38 Oe, respectively. Both theoretical prediction and experimental results show that smaller sensors have greater mass sensitivity and can theoretically detect mass as small as one biological spore. The sensor platform was immobilized with JRB7 phages for specific, in vitro detection of B. anthracis spores. Real-time detection of spores suspended in water was demonstrated using a flowing system. The 1 mm and 2 mm sensors were found to have a detection limit of 104 spores ml-1 and 105 spores ml-1, respectively.

Detect-to-treat: development of analysis of bacilli spores in nasal mucus by surfaced-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Inscore, Frank E.; Gift, Alan D.; Farquharson, Stuart

2004-12-01

As the war on terrorism in Afghanistan and Iraq continue, future attacks both abroad and in the U.S.A. are expected. In an effort to aid civilian and military personnel, we have been investigating the potential of using a surface-enhanced Raman spectroscopy (SERS) sampling device to detect Bacillus anthracis spores in nasal swab samples. Such a device would be extremely beneficial to medical responders and management in assessing the extent of a bioterrorist attack and making detect-to-treat decisions. The disposable sample device consists of a glass capillary filled with a silver-doped sol-gel that is capable of extracting dipicolinic acid (DPA), a chemical signature of Bacilli, and generating SERS spectra. The sampling device and preliminary measurements of DPA extracted from spores and nasal mucus will be presented.

Unlocking the Sporicidal Potential of Ethanol: Induced Sporicidal Activity of Ethanol against Clostridium difficile and Bacillus Spores under Altered Physical and Chemical Conditions

PubMed Central

Nerandzic, Michelle M.; Sunkesula, Venkata C. K.; C., Thriveen Sankar; Setlow, Peter; Donskey, Curtis J.

2015-01-01

Background Due to their efficacy and convenience, alcohol-based hand sanitizers have been widely adopted as the primary method of hand hygiene in healthcare settings. However, alcohols lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We hypothesized that sporicidal activity could be induced in alcohols through alteration of physical or chemical conditions that have been shown to degrade or allow penetration of spore coats. Principal Findings Acidification, alkalinization, and heating of ethanol induced rapid sporicidal activity against C. difficile, and to a lesser extent Bacillus thuringiensis and Bacillus subtilis. The sporicidal activity of acidified ethanol was enhanced by increasing ionic strength and mild elevations in temperature. On skin, sporicidal ethanol formulations were as effective as soap and water hand washing in reducing levels of C. difficile spores. Conclusions These findings demonstrate that novel ethanol-based sporicidal hand hygiene formulations can be developed through alteration of physical and chemical conditions. PMID:26177038

Small acid soluble proteins for rapid spore identification.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.

2006-12-01

This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescencemore » detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.« less

A plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with Bacillus anthracis Ames spores.

PubMed

Chichester, Jessica A; Manceva, Slobodanka D; Rhee, Amy; Coffin, Megan V; Musiychuk, Konstantin; Mett, Vadim; Shamloul, Moneim; Norikane, Joey; Streatfield, Stephen J; Yusibov, Vidadi

2013-03-01

The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our launch vector-based plant transient expression system. Using this system, we have successfully engineered, expressed, purified and characterized full-length PA (pp-PA83) in Nicotiana benthamiana plants using agroinfiltration. This plant-produced antigen elicited high toxin neutralizing antibody titers in mice and rabbits after two vaccine administrations with Alhydrogel. In addition, immunization with this vaccine candidate protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. The vaccine effects were dose-dependent and required the presence of Alhydrogel adjuvant. In addition, the vaccine antigen formulated with Alhydrogel was stable and retained immunogenicity after two-week storage at 4°C, the conditions intended for clinical use. These results support the testing of this vaccine candidate in human volunteers and the utility of our plant expression system for the production of a recombinant anthrax vaccine.

Cutaneous anthrax: an overview.

PubMed

Celia, Frank

2002-04-01

The recent acts of bioterrorism have raised new questions about this uncommon disease. Clinicians are puzzled as to why some of the victims exposed to Bacillus anthracis spores developed the cutaneous form of the disease and others the inhalational form. Despite these questions, cutaneous anthrax remains relatively simple to treat effectively. The real clinical challenge lies in the diagnosis, especially being able to distinguish it from a spider bite.

STRUCTURE OF THE TYPE III PANTOTHENATE KINASE FROM Bacillus anthracis AT 2.0 Å RESOLUTION

PubMed Central

Nicely, Nathan I.; Parsonage, Derek; Paige, Carleitta; Newton, Gerald L.; Fahey, Robert C.; Leonardi, Roberta; Jackowski, Suzanne; Mallett, T. Conn; Claiborne, Al

2008-01-01

Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-11289]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 Å, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen. PMID:17323930

Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk.

PubMed

Tomasula, P M; Mukhopadhyay, S; Datta, N; Porto-Fett, A; Call, J E; Luchansky, J B; Renye, J; Tunick, M

2011-09-01

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log(10) BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91±0.05 log(10) BA spores/mL of milk and the 1.4-μm membrane removed 4.50±0.35 log(10) BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log(10) spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log(10) BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Report for the NGFA-5 project.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaing, C; Jackson, P; Thissen, J

The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, TaqMan PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. To effectively compare the sensitivity and specificity of the different genomic technologies, we used SNP TaqMan PCR, MLVA, microarray and high-throughput illumine and 454 sequencing to test various strains from B. anthracis, B. thuringiensis, BioWatch aerosol filter extracts or soil samples that were spiked with B. anthracis, and samples that were previously collected during DHS and EPAmore » environmental release exercises that were known to contain B. thuringiensis spores. The results of all the samples against the various assays are discussed in this report.« less

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bartholomew, Rachel A.; Ozanich, Richard M.; Arce, Jennifer S.

2017-02-01

The goal of this testing was to evaluate the ability of currently available commercial off-the-shelf (COTS) biological indicator tests and immunoassays to detect Bacillus anthracis (Ba) spores and ricin. In general, immunoassays provide more specific identification of biological threats as compared to indicator tests [3]. Many of these detection products are widely used by first responders and other end users. In most cases, performance data for these instruments are supplied directly from the manufacturer, but have not been verified by an external, independent assessment [1]. Our test plan modules included assessments of inclusivity (ability to generate true positive results), commonlymore » encountered hoax powders (which can cause potential interferences or false positives), and estimation of limit of detection (LOD) (sensitivity) testing.« less

Proteomic signatures differentiating Bacillus anthracis Sterne sporulation on soil relative to laboratory media.

PubMed

Wunschel, D S; Hutchison, J R; Deatherage Kaiser, B L; Merkley, E D; Hess, B M; Lin, A; Warner, M G

2017-12-18

The process of sporulation is vital for the stability and infectious cycle of Bacillus anthracis. The spore is the infectious form of the organism and therefore relevant to biodefense. While the morphological and molecular events occurring during sporulation have been well studied, the influence of growth medium and temperature on the proteins expressed in sporulated cultures is not well understood. Understanding the features of B. anthracis sporulation specific to natural vs. laboratory production will address an important question in microbial forensics. In an effort to bridge this knowledge gap, a system for sporulation on two types of agar-immobilized soils was used for comparison to cultures sporulated on two common types of solid laboratory media, and one liquid sporulation medium. The total number of proteins identified as well as their identity differed between samples generated in each medium and growth temperature, demonstrating that sporulation environment significantly impacts the protein content of the spore. In addition, a subset of proteins common in all of the soil-cultivated samples was distinct from the expression profiles in laboratory medium (and vice versa). These differences included proteins involved in thiamine and phosphate metabolism in the sporulated cultures produced on soils with a notable increase in expression of ATP binding cassette (ABC) transporters annotated to be for phosphate and antimicrobial peptides. A distinct set of ABC transporters for amino acids, sugars and oligopeptides were found in cultures produced on laboratory media as well as increases in carbon and amino acid metabolism-related proteins. These protein expression changes indicate that the sporulation environment impacts the protein profiles in specific ways that are reflected in the metabolic and membrane transporter proteins present in sporulated cultures.

Evaluation of standardized sample collection, packaging, and decontamination procedures to assess cross-contamination potential during Bacillus anthracis incident response operations

PubMed Central

Calfee, M. Worth; Tufts, Jenia; Meyer, Kathryn; McConkey, Katrina; Mickelsen, Leroy; Rose, Laura; Dowell, Chad; Delaney, Lisa; Weber, Angela; Morse, Stephen; Chaitram, Jasmine; Gray, Marshall

2016-01-01

Sample collection procedures and primary receptacle (sample container and bag) decontamination methods should prevent contaminant transfer between contaminated and non-contaminated surfaces and areas during bio-incident operations. Cross-contamination of personnel, equipment, or sample containers may result in the exfiltration of biological agent from the exclusion (hot) zone and have unintended negative consequences on response resources, activities and outcomes. The current study was designed to: (1) evaluate currently recommended sample collection and packaging procedures to identify procedural steps that may increase the likelihood of spore exfiltration or contaminant transfer; (2) evaluate the efficacy of currently recommended primary receptacle decontamination procedures; and (3) evaluate the efficacy of outer packaging decontamination methods. Wet- and dry-deposited fluorescent tracer powder was used in contaminant transfer tests to qualitatively evaluate the currently-recommended sample collection procedures. Bacillus atrophaeus spores, a surrogate for Bacillus anthracis, were used to evaluate the efficacy of spray- and wipe-based decontamination procedures. Both decontamination procedures were quantitatively evaluated on three types of sample packaging materials (corrugated fiberboard, polystyrene foam, and polyethylene plastic), and two contamination mechanisms (wet or dry inoculums). Contaminant transfer results suggested that size-appropriate gloves should be worn by personnel, templates should not be taped to or removed from surfaces, and primary receptacles should be selected carefully. The decontamination tests indicated that wipe-based decontamination procedures may be more effective than spray-based procedures; efficacy was not influenced by material type but was affected by the inoculation method. Incomplete surface decontamination was observed in all tests with dry inoculums. This study provides a foundation for optimizing current B. anthracis response procedures to minimize contaminant exfiltration. PMID:27362274

Historical evolution of human anthrax from occupational disease to potentially global threat as bioweapon.

PubMed

D'Amelio, Enrico; Gentile, Bernardina; Lista, Florigio; D'Amelio, Raffaele

2015-12-01

Anthrax is caused by Bacillus anthracis, which can naturally infect livestock, wildlife and occupationally exposed humans. However, for its resistance due to spore formation, ease of dissemination, persistence in the environment and high virulence, B. anthracis has been considered the most serious bioterrorism agent for a long time. During the last century anthrax evolved from limited natural disease to potentially global threat if used as bioweapon. Several factors may mitigate the consequences of an anthrax attack, including 1. the capability to promptly recognize and manage the illness and its public health consequences; 2. the limitation of secondary contamination risk through an appropriate decontamination; and 3. the evolution of genotyping methods (for microbes characterization at high resolution level) that can influence the course and/or focus of investigations, impacting the response of the government to an attack. A PubMed search has been done using the key words “bioterrorism anthrax”. Over one thousand papers have been screened and the most significant examined to present a comprehensive literature review in order to discuss the current knowledge and strategies in preparedness for a possible deliberate release of B. anthracis spores and to indicate the most current and complete documents in which to deepen. The comprehensive analysis of the two most relevant unnatural anthrax release events, Sverdlovsk in the former Soviet Union (1979) and the contaminated letters in the USA (2001), shows that inhalational anthrax may easily and cheaply be spread resulting in serious consequences. The damage caused by an anthrax attack can be limited if public health organization, first responders, researchers and investigators will be able to promptly manage anthrax cases and use new technologies for decontamination methods and in forensic microbiology.

Evaluation of standardized sample collection, packaging, and decontamination procedures to assess cross-contamination potential during Bacillus anthracis incident response operations.

PubMed

Calfee, M Worth; Tufts, Jenia; Meyer, Kathryn; McConkey, Katrina; Mickelsen, Leroy; Rose, Laura; Dowell, Chad; Delaney, Lisa; Weber, Angela; Morse, Stephen; Chaitram, Jasmine; Gray, Marshall

2016-12-01

Sample collection procedures and primary receptacle (sample container and bag) decontamination methods should prevent contaminant transfer between contaminated and non-contaminated surfaces and areas during bio-incident operations. Cross-contamination of personnel, equipment, or sample containers may result in the exfiltration of biological agent from the exclusion (hot) zone and have unintended negative consequences on response resources, activities and outcomes. The current study was designed to: (1) evaluate currently recommended sample collection and packaging procedures to identify procedural steps that may increase the likelihood of spore exfiltration or contaminant transfer; (2) evaluate the efficacy of currently recommended primary receptacle decontamination procedures; and (3) evaluate the efficacy of outer packaging decontamination methods. Wet- and dry-deposited fluorescent tracer powder was used in contaminant transfer tests to qualitatively evaluate the currently-recommended sample collection procedures. Bacillus atrophaeus spores, a surrogate for Bacillus anthracis, were used to evaluate the efficacy of spray- and wipe-based decontamination procedures. Both decontamination procedures were quantitatively evaluated on three types of sample packaging materials (corrugated fiberboard, polystyrene foam, and polyethylene plastic), and two contamination mechanisms (wet or dry inoculums). Contaminant transfer results suggested that size-appropriate gloves should be worn by personnel, templates should not be taped to or removed from surfaces, and primary receptacles should be selected carefully. The decontamination tests indicated that wipe-based decontamination procedures may be more effective than spray-based procedures; efficacy was not influenced by material type but was affected by the inoculation method. Incomplete surface decontamination was observed in all tests with dry inoculums. This study provides a foundation for optimizing current B. anthracis response procedures to minimize contaminant exfiltration.

Evaluation of immunogenicity and efficacy of anthrax vaccine adsorbed for postexposure prophylaxis.

PubMed

Ionin, Boris; Hopkins, Robert J; Pleune, Brett; Sivko, Gloria S; Reid, Frances M; Clement, Kristin H; Rudge, Thomas L; Stark, Gregory V; Innes, Alison; Sari, Suha; Guina, Tina; Howard, Cris; Smith, Jeffrey; Swoboda, M Lisa; Vert-Wong, Ekaterina; Johnson, Virginia; Nabors, Gary S; Skiadopoulos, Mario H

2013-07-01

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.

Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax.

PubMed

Oscherwitz, Jon; Feldman, Daniel; Yu, Fen; Cease, Kemp B

2015-01-09

Anthrax represents a formidable bioterrorism threat for which new, optimized vaccines are required. We previously demonstrated that epitope-focused multiple antigenic peptides or a recombinant protein in Freund's adjuvant can elicit Ab against the loop neutralizing determinant (LND), a cryptic linear neutralizing epitope in the 2ß2-2ß3 loop of protective antigen from Bacillus anthracis, which mediated protection of rabbits from inhalation challenge with B. anthracis Ames strain. However, demonstration of efficacy using human-use adjuvants is required before proceeding with further development of an LND vaccine for testing in non-human primates and humans. To optimize the LND immunogen, we first evaluated the protective efficacy and immune correlates associated with immunization of rabbits with mixtures containing two molecular variants of multiple antigenic peptides in Freunds adjuvant, termed BT-LND(2) and TB-LND(2). TB-LND(2) was then further evaluated for protective efficacy in rabbits employing human-use adjuvants. Immunization of rabbits with TB-LND(2) in human-use adjuvants elicited protection from Ames strain spore challenge which was statistically indistinguishable from that elicited through immunization with protective antigen. All TB-LND(2) rabbits with any detectable serum neutralization prior to challenge were protected from aerosolized spore exposure. Remarkably, rabbits immunized with TB-LND(2) in Alhydrogel/CpG had significant anamnestic increases in post-challenge LND-specific Ab and neutralization titers despite little evidence of spore germination in these rabbits. An LND-specific epitope-focused vaccine may complement PA-based vaccines and may represent a complementary stand-alone vaccine for anthrax. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

PubMed

Yang, Yu; Wang, Jing; Wen, Haiyan; Liu, Hengchuan

2012-01-01

We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

PubMed

Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

2015-01-01

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

Full Scale Drinking Water System Decontamination at the Water Security Test Bed.

PubMed

Szabo, Jeffrey; Hall, John; Reese, Steve; Goodrich, Jim; Panguluri, Sri; Meiners, Greg; Ernst, Hiba

2018-03-20

The EPA's Water Security Test Bed (WSTB) facility is a full-scale representation of a drinking water distribution system. In collaboration with the Idaho National Laboratory (INL), EPA designed the WSTB facility to support full-scale evaluations of water infrastructure decontamination, real-time sensors, mobile water treatment systems, and decontamination of premise plumbing and appliances. The EPA research focused on decontamination of 1) Bacillus globigii (BG) spores, a non-pathogenic surrogate for Bacillus anthracis and 2) Bakken crude oil. Flushing and chlorination effectively removed most BG spores from the bulk water but BG spores still remained on the pipe wall coupons. Soluble oil components of Bakken crude oil were removed by flushing although oil components persisted in the dishwasher and refrigerator water dispenser. Using this full-scale distribution system allows EPA to 1) test contaminants without any human health or ecological risk and 2) inform water systems on effective methodologies responding to possible contamination incidents.

Surface Sampling of Spores in Dry-Deposition Aerosols▿

PubMed Central

Edmonds, Jason M.; Collett, Patricia J.; Valdes, Erica R.; Skowronski, Evan W.; Pellar, Gregory J.; Emanuel, Peter A.

2009-01-01

The ability to reliably and reproducibly sample surfaces contaminated with a biological agent is a critical step in measuring the extent of contamination and determining if decontamination steps have been successful. The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated by the fact that no standard sample collection format or decontamination procedures were established. Recovery efficiencies traditionally have been calculated based upon biological agents which were applied to test surfaces in a liquid format and then allowed to dry prior to sampling tests, which may not be best suited for a real-world event with aerosolized biological agents. In order to ascertain if differences existed between air-dried liquid deposition and biological spores which were allowed to settle on a surface in a dried format, a study was undertaken to determine if differences existed in surface sampling recovery efficiencies for four representative surfaces. Studies were then undertaken to compare sampling efficiencies between liquid spore deposition and aerosolized spores which were allowed to gradually settle under gravity on four different test coupon types. Tests with both types of deposition compared efficiencies of four unique swabbing materials applied to four surfaces with various surface properties. Our studies demonstrate that recovery of liquid-deposited spores differs significantly from recovery of dry aerosol-deposited spores in most instances. Whether the recovery of liquid-deposited spores is overexaggerated or underrepresented with respect to that of aerosol-deposited spores depends upon the surface material being tested. PMID:18997021

Feasibility of Wide-Area Decontamination of Bacillus anthracis Spores Using a Germination-Lysis Approach

DTIC Science & Technology

2011-11-16

Security, LLC 2011 CBD S& T Conference November 16, 2011 LLNL-PRES-508394 Lawrence Livermore National Laboratory LLNL-PRES-  Background...PRES-  Gruinard Island 5% formaldehyde  Sverdlosk Release UNKNOWN: but washing, chloramines , soil disposal believed to have been used...508394 Lawrence Livermore National Laboratory LLNL-PRES- 4 Disinfectant >6 Log Reduction on Materials (EPA, 2010a,b; Wood et al., 2011

A retrospective study of anthrax on the Ghaap Plateau, Northern Cape province of South Africa, with special reference to the 2007-2008 outbreaks.

PubMed

Hassim, Ayesha; Dekker, Edgar H; Byaruhanga, Charles; Reardon, Tommy; Van Heerden, Henriette

2017-09-28

Anthrax is a zoonotic disease caused by the gram-positive, endospore-forming and soil-borne bacterium Bacillus anthracis. When in spore form, the organism can survive in dormancy in the environment for decades. It is a controlled disease of livestock and wild ungulates in South Africa. In South Africa, the two enzootic regions are the Kruger National Park and the Ghaap Plateau in the Northern Cape province. Farms on the Plateau span thousands of hectares comprising of wildlife - livestock mixed use farming. In 2007-2008, anthrax outbreaks in the province led to government officials intervening to aid farmers with control measures aimed at preventing further losses. Because of the ability of the organism to persist in the environment for prolonged periods, an environmental risk or isolation survey was carried out in 2012 to determine the efficacy of control measures employed during the 2007-2008, anthrax outbreaks. No B. anthracis could be isolated from the old carcass sites, even when bone fragments from the carcasses were still clearly evident. This is an indication that the control measures and protocols were apparently successful in stemming the continuity of spore deposits at previously positive carcass sites.

Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

NASA Technical Reports Server (NTRS)

Kminek, Gerhard; Bada, Jeffrey L.; Pogliano, Kit; Ward, John F.

2003-01-01

When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and s. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old, B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus. To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the upper-most meter of Mars.

National validation study of a swab protocol for the recovery of Bacillus anthracis spores from surfaces.

PubMed

Hodges, Lisa R; Rose, Laura J; O'Connell, Heather; Arduino, Matthew J

2010-05-01

Twelve Laboratory Response Network (LRN) affiliated laboratories participated in a validation study of a macrofoam swab protocol for the recovery, detection, and quantification of viable B. anthracis (BA) Sterne spores from steel surfaces. CDC personnel inoculated steel coupons (26cm(2)) with 1-4 log(10) BA spores and recovered them by sampling with pre-moistened macrofoam swabs. Phase 1 (P1) of the study evaluated swabs containing BA only, while dust and background organisms were added to swabs in Phase 2 (P2) to mimic environmental conditions. Laboratories processed swabs and enumerated spores by culturing eluted swab suspensions and counting colonies with morphology consistent with BA. Processed swabs were placed in enrichment broth, incubated 24h, and cultured by streaking for isolation. Real-time PCR was performed on selected colonies from P2 samples to confirm the identity of BA. Mean percent recovery (%R) of spores from the surface ranged from 15.8 to 31.0% (P1) and from 27.9 to 55.0% (P2). The highest mean percent recovery was 31.0% (sd 10.9%) for P1 (4 log(10) inoculum) and 55.0% (sd 27.6%) for P2 (1 log(10) inoculum). The overall %R was higher for P2 (44.6%) than P1 (24.1%), but the overall reproducibility (between-lab variability) was lower in P2 than in P1 (25.0 vs 16.5%CV, respectively). The overall precision (within-lab variability) was close to identical for P1 and P2 (44.0 and 44.1, respectively), but varied greatly between inoculum levels. The protocol demonstrated linearity in %R over the three inoculum levels and is able to detect between 26 and 5x10(6)spores/26cm(2). Sensitivity as determined by culture was >98.3% for both phases and all inocula, suggesting that the culture method maintains sensitivity in the presence of contaminants. The enrichment broth method alone was less sensitive for sampled swabs (66.4%) during P2, suggesting that the presence of background organisms inhibited growth or isolation of BA from the broth. The addition of real-time PCR testing to the assay increased specificity from >85.4% to >95.0% in P2. Although the precision was low at the 1 log(10) inoculum level in both phases (59.0 and 50.2%), this swab processing protocol, was sensitive, specific, precise, and reproducible at 2-4 log(10)/26cm(2) spore concentrations. Published by Elsevier B.V.

Experimental Design for a Macrofoam Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piepel, Gregory F.; Hutchison, Janine R.

2014-04-16

This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (plating/counting and polymerase chain reaction) will be used. Only one previous study has investigated false negative as a function of affecting test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completingmore » gaps in the available information on the performance of macrofoam swab sampling at low concentrations.« less

Experimental Design for a Macrofoam-Swab Study Relating the Recovery Efficiency and False Negative Rate to Low Concentrations of Two Bacillus anthracis Surrogates on Four Surface Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piepel, Gregory F.; Hutchison, Janine R.

This report describes the experimental design for a laboratory study to quantify the recovery efficiencies and false negative rates of a validated, macrofoam-swab sampling method for low concentrations of Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus (BG) spores on four surface materials (stainless steel, glass, vinyl tile, plastic light cover panel). Two analytical methods (culture and polymerase chain reaction) will be used. Only one previous study has investigated how the false negative rate depends on test factors. The surrogates BAS and BG have not been tested together in the same study previously. Hence, this study will provide for completing gapsmore » in the available information on the performance of macrofoam-swab sampling at low concentrations.« less

Decontamination of biological warfare agents by a microwave plasma torch

NASA Astrophysics Data System (ADS)

Lai, Wilson; Lai, Henry; Kuo, Spencer P.; Tarasenko, Olga; Levon, Kalle

2005-02-01

A portable arc-seeded microwave plasma torch running stably with airflow is described and applied for the decontamination of biological warfare agents. Emission spectroscopy of the plasma torch indicated that this torch produced an abundance of reactive atomic oxygen that could effectively oxidize biological agents. Bacillus cereus was chosen as a simulant of Bacillus anthracis spores for biological agent in the decontamination experiments. Decontamination was performed with the airflow rate of 0.393l/s, corresponding to a maximum concentration of atomic oxygen produced by the torch. The experimental results showed that all spores were killed in less than 8 s at 3 cm distance, 12 s at 4 cm distance, and 16 s at 5 cm distance away from the nozzle of the torch.

Phase 1 Testing of Bioflash Technology for White Powder Identification

DTIC Science & Technology

2012-06-01

limit of detection for the test bed system for powdered spores of Bacillus anthracis and Bacillus subtilis , and (2) to determine if common nonhazardous... Bacillus subtilis ; and (2) if common nonhazardous white powders trigger a false positive response or subsequently interfere with the ability of the...Isolated from Flour and Ropy Bread. Letters in App Microbiol. 2003, 37, 169-173. Te Giffel, M.C. Incidence of Bacillus cereus and Bacillus subtilis in

Thermal Inactivation of Bacillus Anthracis using Laser Irradiation of Micro-Etched Platforms

DTIC Science & Technology

2009-03-01

Application of Malachite Green Staining protocol for greater clarity of spore population; fluorescent staining techniques. Further assess toxicity or...to normal staining procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells with heat. Malachite ...dimethyl-aniline is a toxic chemical primarily used as a dye. Since malachite green is water-soluble and does not adhere well to the cell, and since the

Detergent-Resistant Membrane Microdomains Facilitate Ib Oligomer Formation and Biological Activity of Clostridium perfringens Iota-Toxin

DTIC Science & Technology

2004-04-01

spore-forming bacilli such as Clostridium spiroforme (iota-like toxin), Clostridium botulinum (C2 toxin), Bacillus anthracis (lethal and edema toxins...ously (28). Goat C. spiroforme and C. perfringens type C antisera were purchased from TechLab, Inc. (Blacksburg, Va.). Mouse monoclonal antibodies...membrane preparations was specific. Previous studies showed that the binary C. spiroforme toxin shares common epitopes with iota-toxin, and antisera

Decontamination of Subway Infrastructure Materials ...

EPA Pesticide Factsheets

Report This report provides the results of an assessment to determine the decontamination efficacy of methyl bromide (MB) fumigant in inactivating Bacillus anthracis (B.a.; causative agent for anthrax) spores on materials typically found in subway system infrastructure. To facilitate future decontaminations employing MB in a subway environment, this investigation focused on finding efficacious conditions when using MB at temperatures that may be encountered in an underground subway system (i.e., temperatures lower than used in previous studies).

False-negative rate, limit of detection and recovery efficiency performance of a validated macrofoam-swab sampling method for low surface concentrations of Bacillus anthracis Sterne and Bacillus atrophaeus spores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piepel, G. F.; Deatherage Kaiser, B. L.; Amidan, B. G.

The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » vinyl tile (50.8% with BAS and 40.2% with BG) and the highest for glass (92.8% with BAS and 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG; values increased as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent article.« less

Composite Sampling Approaches for Bacillus anthracis Surrogate Extracted from Soil

PubMed Central

France, Brian; Bell, William; Chang, Emily; Scholten, Trudy

2015-01-01

Any release of anthrax spores in the U.S. would require action to decontaminate the site and restore its use and operations as rapidly as possible. The remediation activity would require environmental sampling, both initially to determine the extent of contamination (hazard mapping) and post-decon to determine that the site is free of contamination (clearance sampling). Whether the spore contamination is within a building or outdoors, collecting and analyzing what could be thousands of samples can become the factor that limits the pace of restoring operations. To address this sampling and analysis bottleneck and decrease the time needed to recover from an anthrax contamination event, this study investigates the use of composite sampling. Pooling or compositing of samples is an established technique to reduce the number of analyses required, and its use for anthrax spore sampling has recently been investigated. However, use of composite sampling in an anthrax spore remediation event will require well-documented and accepted methods. In particular, previous composite sampling studies have focused on sampling from hard surfaces; data on soil sampling are required to extend the procedure to outdoor use. Further, we must consider whether combining liquid samples, thus increasing the volume, lowers the sensitivity of detection and produces false negatives. In this study, methods to composite bacterial spore samples from soil are demonstrated. B. subtilis spore suspensions were used as a surrogate for anthrax spores. Two soils (Arizona Test Dust and sterilized potting soil) were contaminated and spore recovery with composites was shown to match individual sample performance. Results show that dilution can be overcome by concentrating bacterial spores using standard filtration methods. This study shows that composite sampling can be a viable method of pooling samples to reduce the number of analysis that must be performed during anthrax spore remediation. PMID:26714315

Bacillus anthracis Capsular Conjugates Elicit Chimpanzee Polyclonal Antibodies That Protect Mice from Pulmonary Anthrax.

PubMed

Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie A; Dai, Zhongdong; Kubler-Kielb, Joanna; Agulto, Liane; Leppla, Stephen H; Purcell, Robert H

2015-08-01

The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid [PGA]) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above 10(4) following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis. Most important, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as expanded-spectrum anthrax vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramosa.

PubMed

Mouton, Laurence; Traunecker, Emmanuel; McElroy, Kerensa; Du Pasquier, Louis; Ebert, Dieter

2009-12-01

Pasteuria ramosa is a spore-forming bacterium that infects Daphnia species. Previous results demonstrated a high specificity of host clone/parasite genotype interactions. Surface proteins of bacteria often play an important role in attachment to host cells prior to infection. We analyzed surface proteins of P. ramosa spores by two-dimensional gel electrophoresis. For the first time, we prove that two isolates selected for their differences in infectivity reveal few but clear-cut differences in protein patterns. Using internal sequencing and LC/MS/MS, we identified a collagen-like protein named Pcl1a (Pasteuria collagen-like protein 1a). This protein, reconstructed with the help of Pasteuria genome sequences, contains three domains: a 75-amino-acid amino-terminal domain with a potential transmembrane helix domain, a central collagen-like region (CLR) containing Gly-Xaa-Yaa (GXY) repeats, and a 7-amino-acid carboxy-terminal domain. The CLR region is polymorphic among the two isolates with amino-acid substitutions and a variable number of GXY triplets. Collagen-like proteins are rare in prokaryotes, although they have been described in several pathogenic bacteria, including Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, closely related to Pasteuria species, in which they could be involved in the adherence of bacteria to host cells.

Decontamination of Drinking Water Infrastructure ...

EPA Pesticide Factsheets

Technical Brief This study examines the effectiveness of decontaminating corroded iron and cement-mortar coupons that have been contaminated with spores of Bacillus atrophaeus subsp. globigii (B. globigii), which is often used as a surrogate for pathogenic B. anthracis (anthrax) in disinfection studies. Bacillus spores are persistent on common drinking water material surfaces like corroded iron, requiring physical or chemical methods to decontaminate the infrastructure. In the United States, free chlorine and monochloramine are the primary chemical disinfectants used by the drinking water industry to inactivate microorganisms. Flushing is also a common, easily implemented practice in drinking water distribution systems, although large volumes of contaminated water needing treatment could be generated. Identifying readily available alternative disinfectant formulations for infrastructure decontamination could give water utilities options for responding to specific types of contamination events. In addition to presenting data on flushing alone, which demonstrated the persistence of spores on water infrastructure in the absence of high levels of disinfectants, data on acidified nitrite, chlorine dioxide, free chlorine, monochloramine, ozone, peracetic acid, and followed by flushing are provided.

Anthrax vaccines: present status and future prospects.

PubMed

Kaur, Manpreet; Singh, Samer; Bhatnagar, Rakesh

2013-08-01

The management of anthrax remains a top priority among the biowarfare/bioterror agents. It was the Bacillus anthracis spore attack through the US mail system after the September 11, 2001, terrorist attacks in the USA that highlighted the potential of B. anthracis as a bioterrorism agent and the threat posed by its deliberate dissemination. These attacks invigorated the efforts toward understanding the anthrax pathogenesis and development of more comprehensive medical intervention strategies for its containment in case of both natural disease and manmade, accidental or deliberate infection of a non-suspecting population. Currently, efforts are directed toward the development of safe and efficacious vaccines as well as intervention tools for controlling the disease in the advanced fulminant stage when toxemia has already developed. This work presents an overview of the current understanding of anthrax pathogenesis and recent advances made, particularly after 2001, for the successful management of anthrax and outlines future perspectives.

Medical toxicology and public health-update on research and activities at the centers for disease control and prevention and the agency for toxic substances and disease registry.

PubMed

Snook, Curtis P; Cardarelli, John; Mickelsen, R Leroy; Mattorano, Dino; Nalipinski, Michael

2008-12-01

An extensive review of CDC epidemiological responses to human outbreaks of anthrax from occupational settings between the years of 1950 and 2001 documented a variety of approaches to mitigation and decontamination [2]. These approaches included taking no action, burning contaminated materials, chlorinating water supplies, instituting administrative and engineering controls and PPE, vaccinating potentially exposed individuals, and in 2 instances, fumigating with formaldehyde vapor (now considered to be a human carcinogen). Secondary contamination of a worker's home was documented in 1 case, but not felt to be clinically significant to warrant any decontamination efforts. In response to the B. anthracis attacks in 2001, chlorine dioxide fumigation, vaporous hydrogen peroxide fumigation, and a combination of HEPA vacuuming, cleaning, and bleach application were all techniques used successfully to clean B. anthracis spore contamination.

Measuring the Variability of Treated Bacillus Anthracis Delta Stern Spores

DTIC Science & Technology

2011-02-23

Edgewood, MD) and 107 grown in nine 4L cultures of G-media plus trace metals at 37 o C until >95% sporulated . Percent 108 sporulation was determined...1 (not shown). 385 Sporulated bacteria have long been known to be more resilient (4) than vegetative forms. Left-386 over, unaccounted for, dead...growth treatment of sporulated test 487 materials are needed to bring the use of biological test materials into a uniform quality system. 488 489

Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

PubMed

Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

2016-02-25

Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R.

Petrobactin Is Exported from Bacillus anthracis by the RND-Type Exporter ApeX

PubMed Central

Hagan, A. K.; Berger, D.

2017-01-01

ABSTRACT Bacillus anthracis—a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cell. However, growth of ΔapeX mutants in iron-depleted medium was not affected, and virulence in mice was not attenuated. Instead, petrobactin components were determined to be exported through a different protein, which enables iron transport sufficient for growth, albeit with a slightly lower affinity for iron. This is the first report to identify a functional siderophore exporter in B. anthracis and the in vivo functionality of siderophore components. Moreover, this is the first application of LAESI-MS to sample a virulence factor/metabolite directly from bacterial culture media and cell pellets of a human pathogen. PMID:28900020

Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong-geller, Elizabeth; Valdez, Yolanda E; Shou, Yulin

2009-01-01

Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs ormore » wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.« less

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real-time PCR.

PubMed

Hong-Geller, E; Valdez, Y E; Shou, Y; Yoshida, T M; Marrone, B L; Dunbar, J M

2010-04-01

We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. We evaluated the sample recovery efficiencies of two collection methods - swabs and wipes - for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

Effectiveness of Spray-Based Decontamination Methods for ...

EPA Pesticide Factsheets

Report The objective of this project was to assess the effectiveness of spray-based common decontamination methods for inactivating Bacillus (B.) atrophaeus (surrogate for B. anthracis) spores and bacteriophage MS2 (surrogate for foot and mouth disease virus [FMDV]) on selected test surfaces (with or without a model agricultural soil load). Relocation of viable viruses or spores from the contaminated coupon surfaces into aerosol or liquid fractions during the decontamination methods was investigated. This project was conducted to support jointly held missions of the U.S. Department of Homeland Security (DHS) and the U.S. Environmental Protection Agency (EPA). Within the EPA, the project supports the mission of EPA’s Homeland Security Research Program (HSRP) by providing relevant information pertinent to the decontamination of contaminated areas resulting from a biological incident.

Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

PubMed Central

Fan, Maomian; McBurnett, Shelly Roper; Andrews, Carrie J.; Allman, Amity M.; Bruno, John G.; Kiel, Johnathan L.

2008-01-01

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system. PMID:19183794

The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Bacillus anthracis Infection in BALB/c Mice and Cynomolgus Macaques.

PubMed

Grossman, Trudy H; Anderson, Michael S; Drabek, Lindsay; Gooldy, Melanie; Heine, Henry S; Henning, Lisa N; Lin, Winston; Newman, Joseph V; Nevarez, Rene; Siefkas-Patterson, Kaylyn; Radcliff, Anne K; Sutcliffe, Joyce A

2017-10-01

The fluorocycline TP-271 was evaluated in mouse and nonhuman primate (NHP) models of inhalational anthrax. BALB/c mice were exposed by nose-only aerosol to Bacillus anthracis Ames spores at a level of 18 to 88 lethal doses sufficient to kill 50% of exposed individuals (LD 50 ). When 21 days of once-daily dosing was initiated at 24 h postchallenge (the postexposure prophylaxis [PEP] study), the rates of survival for the groups treated with TP-271 at 3, 6, 12, and 18 mg/kg of body weight were 90%, 95%, 95%, and 84%, respectively. When 21 days of dosing was initiated at 48 h postchallenge (the treatment [Tx] study), the rates of survival for the groups treated with TP-271 at 6, 12, and 18 mg/kg TP-271 were 100%, 91%, and 81%, respectively. No deaths of TP-271-treated mice occurred during the 39-day posttreatment observation period. In the NHP model, cynomolgus macaques received an average dose of 197 LD 50 of B. anthracis Ames spore equivalents using a head-only inhalation exposure chamber, and once-daily treatment of 1 mg/kg TP-271 lasting for 14 or 21 days was initiated within 3 h of detection of protective antigen (PA) in the blood. No (0/8) animals in the vehicle control-treated group survived, whereas all 8 infected macaques treated for 21 days and 4 of 6 macaques in the 14-day treatment group survived to the end of the study (56 days postchallenge). All survivors developed toxin-neutralizing and anti-PA IgG antibodies, indicating an immunologic response. On the basis of the results obtained with the mouse and NHP models, TP-271 shows promise as a countermeasure for the treatment of inhalational anthrax. Copyright © 2017 American Society for Microbiology.

The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Bacillus anthracis Infection in BALB/c Mice and Cynomolgus Macaques

PubMed Central

Anderson, Michael S.; Drabek, Lindsay; Gooldy, Melanie; Heine, Henry S.; Henning, Lisa N.; Lin, Winston; Newman, Joseph V.; Nevarez, Rene; Siefkas-Patterson, Kaylyn; Radcliff, Anne K.; Sutcliffe, Joyce A.

2017-01-01

ABSTRACT The fluorocycline TP-271 was evaluated in mouse and nonhuman primate (NHP) models of inhalational anthrax. BALB/c mice were exposed by nose-only aerosol to Bacillus anthracis Ames spores at a level of 18 to 88 lethal doses sufficient to kill 50% of exposed individuals (LD50). When 21 days of once-daily dosing was initiated at 24 h postchallenge (the postexposure prophylaxis [PEP] study), the rates of survival for the groups treated with TP-271 at 3, 6, 12, and 18 mg/kg of body weight were 90%, 95%, 95%, and 84%, respectively. When 21 days of dosing was initiated at 48 h postchallenge (the treatment [Tx] study), the rates of survival for the groups treated with TP-271 at 6, 12, and 18 mg/kg TP-271 were 100%, 91%, and 81%, respectively. No deaths of TP-271-treated mice occurred during the 39-day posttreatment observation period. In the NHP model, cynomolgus macaques received an average dose of 197 LD50 of B. anthracis Ames spore equivalents using a head-only inhalation exposure chamber, and once-daily treatment of 1 mg/kg TP-271 lasting for 14 or 21 days was initiated within 3 h of detection of protective antigen (PA) in the blood. No (0/8) animals in the vehicle control-treated group survived, whereas all 8 infected macaques treated for 21 days and 4 of 6 macaques in the 14-day treatment group survived to the end of the study (56 days postchallenge). All survivors developed toxin-neutralizing and anti-PA IgG antibodies, indicating an immunologic response. On the basis of the results obtained with the mouse and NHP models, TP-271 shows promise as a countermeasure for the treatment of inhalational anthrax. PMID:28784679

Sampling and inactivation of wet disseminated spores from flooring materials using commercially available robotic vacuum cleaners.

PubMed

Thompson, Katy-Anne; Paton, Susan; Pottage, Thomas; Bennett, Allan

2018-05-09

Four commercially available robotic vacuum cleaners were assessed for sampling efficiency of wet disseminated Bacillus atrophaeus spores on carpet, Polyvinyl Chloride (PVC) and laminate flooring. Furthermore, their operability was evaluated and decontamination efficiency of one robot was assessed using a sodium hypochlorite solution. In an environmental chamber, robots self-navigated around 4 m 2 of flooring containing a single contaminated 0.25 m 2 tile (ca. 10 4 spores per cm 2 ). Contamination levels at pre-determined locations were assessed by macrofoam swabs (PVC and laminate) or water soluble tape (carpet), before and after sampling. Robots were dismantled post-sampling and spore recoveries assessed. Aerosol contamination was also measured during sampling. Robot sampling efficiencies were variable, however, robots recovered most spores from laminate (up to 17.1%), then PVC, and lastly carpet. All robots spread contamination from the 'hotspot' (all robots spread < 0.6% of the contamination to other areas) and became surface contaminated. Spores were detected at low levels during air sampling (<5.6 spores l -1 ). Liquid decontamination inactivated 99.1% of spores from PVC. Robotic vacuum cleaners show promise for both sampling and initial decontamination of indoor flooring. In the event of a bioterror incident, e.g. deliberate release of Bacillus anthracis spores, areas require sampling to determine the magnitude and extent of contamination, and to establish decontamination efficacy. In this study we investigate robotic sampling methods against high concentrations of bacterial spores applied by wet deposition to different floorings, contamination spread to other areas, potential transfer of spores to the operators and assessment of a wet vacuum robot for spore inactivation. The robots' usability was evaluated and how they can be employed in real life scenarios. This will help to reduce the economic cost of sampling and the risk to sampling/decontamination teams. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Functional and Immunological Analyses of Superoxide Dismutases and Other Spore-Associated Proteins of Bacillus anthracis

DTIC Science & Technology

2008-08-20

are strictly zoonotic in origin (with the exception of intentional, bioterror-releated cases) as they result from contact with infected animals or...blocked overnight at 4ºC in PBST (0.5% Tween-20) with 5% nonfat dry milk (PBSTM). To verify the presence of the recombinant protein, blots were then...Whatman GmbH, Dassel, GE), and blocked overnight at 4ºC in PBST (PBS with 0.5% Tween-20) with 5% nonfat dry milk (PBSTM). Purified His- tagged

Efficacy of Oritavancin in a Murine Model of Bacillus anthracis Spore Inhalation Anthrax

DTIC Science & Technology

2008-06-21

Bélanger, and Adel Rafai Far for characterizing oritavancin pharmacokinetics in mice. We acknowledge the support of Ingrid Sarmiento for assistance...antibiotics. FEMS Microbiol. Rev. 26:511–532. 2. Arhin, F. F., I. Sarmiento , A. Belley, G. A. McKay, D. C. Draghi, P. Grover, D. Sahm, T. R. Parr, Jr...Chemother. 52:1597–1603. 3. Arhin, F. F., I. Sarmiento , T. R. Parr, Jr., and G. Moeck. 2007. Mechanisms of action of oritavancin in Staphylococcus

Novel Therapeutic and Prophylactic Modalities to Protect the United States Armed Forces Against Mayor Biological Threat Agents

DTIC Science & Technology

2007-08-01

to protect mice from challenges with purified proteases or B . anthracis spores. k . Generate polyclonal antibodies against recombinant proteases...Leppla, S. H., Agrawa,l S., Jabbar, A., Quinn, C., and Pulendran, B . (2003) Nature 424, 329- 334. 5 Pickering, A. K ., Osorio, M., Lee, G. M., Grippe, V...Douglas, C. M., Patel, S. B ., Wisniewski, D., Scapin, G., Salowe, S. P., Zaller, D. M., Chapman, K . T., Scolnick, E. M., Schmatz, D. M., Bartizal, K

Processing, Assembly and Localization of a Bacillus anthracis Spore Protein

DTIC Science & Technology

2010-01-01

phage transduction, using the CP51 phage as described by Thorne (1968). All mutations were confirmed by PCR analysis (Supplementary Table S1). Protein...with End-It (Epicentre) and self-ligated, creating pKH-KSM4. The region between the T7 terminator and T7 promoter of pET23A (EMD Table 1. Strains and...represent full-length BxpA, we analysed the electrophoretic behaviour of a full-length, histidine-tagged and T7 -tagged version of BxpA overproduced in E

Detection of bacterial endospores by means of ultrafast coherent Raman spectroscopy

NASA Astrophysics Data System (ADS)

Pestov, Dmitry Sergeyevich

This work is devoted to formulation and development of a laser spectroscopic technique for rapid detection of biohazards, such as Bacillus anthracis spores. Coherent anti-Stokes Raman scattering (CARS) is used as an underlying process for active retrieval of species-specific characteristics of an analyte. Vibrational modes of constituent molecules are Raman-excited by a pair of ultrashort, femtosecond laser pulses, and then probed through inelastic scattering of a third, time-delayed laser field. We first employ the already known time-resolved CARS technique. We apply it to the spectroscopy of easy-to-handle methanol-water mixtures, and then continue building our expertise on solutions of dipicolinic acid (DPA) and its salts, which happen to be marker molecules for bacterial spores. Various acquisition schemes are evaluated, and the preference is given to multi-channel frequency-resolved detection, when the whole CARS spectrum is recorded as a function of the probe pulse delay. We demonstrate a simple detection algorithm that manages to differentiate DPA solution from common interferents. We investigate experimentally the advantages and disadvantages of near-resonant probing of the excited molecular coherence, and finally observe the indicative backscattered CARS signal from DPA and NaDPA powders. The possibility of selective Raman excitation via pulse shaping of the preparation pulses is also demonstrated. The analysis of time-resolved CARS experiments on powders and B. subtilis spores, a harmless surrogate for B. anthracis, facilitates the formulation of a new approach, where we take full advantage of the multi-channel frequency-resolved acquisition and spectrally discriminate the Raman-resonant CARS signal from the background due to other instantaneous four-wave mixing (FWM) processes. Using narrowband probing, we decrease the magnitude of the nonresonant FWM, which is further suppressed by the timing of the laser pulses. The devised technique, referred to as hybrid CARS, leads to a single-shot detection of as few as 104 bacterial spores, bringing CARS spectroscopy to the forefront of potential candidates for real-time biohazard detection. It also gives promise to many other applications of CARS, hindered so far by the presence of the overwhelming nonresonant FWM background, mentioned above.

Hot, humid air decontamination of a C-130 aircraft contaminated with spores of two acrystalliferous Bacillus thuringiensis strains, surrogates for Bacillus anthracis.

PubMed

Buhr, T L; Young, A A; Bensman, M; Minter, Z A; Kennihan, N L; Johnson, C A; Bohmke, M D; Borgers-Klonkowski, E; Osborn, E B; Avila, S D; Theys, A M G; Jackson, P J

2016-04-01

To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry(-) HD-1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C-130 aircraft. Bacillus thuringiensis spores were either pre-inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C-130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8-10 log10 m(-2) through the entire cargo hold. After hot, humid air decontamination at 75-80°C, 70-90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m(2) , showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m(-2) . Laboratory test methods for hot, humid air decontamination were scaled for a large-scale aircraft field test. The C-130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft. Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys.

PubMed

Chabot, Donald J; Joyce, Joseph; Caulfield, Michael; Cook, James; Hepler, Robert; Wang, Su; Vietri, Nicholas J; Ruthel, Gordon; Shoop, Wesley; Pitt, Louise; Leffel, Elizabeth; Ribot, Wilson; Friedlander, Arthur M

2012-01-20

Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-γ-D-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines. Published by Elsevier Ltd.

Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars

PubMed Central

Kminek, Gerhard; Bada, Jeffrey L.; Pogliano, Kit; Ward, John F.

2014-01-01

Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003). When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars. PMID:12751954

Pulsed excitation system to measure the resonant frequency of magnetoelastic biosensors

NASA Astrophysics Data System (ADS)

Xie, Hong; Chai, Yating; Horikawa, Shin; Wikle, Howard C.; Chin, Bryan A.

2014-05-01

An electrical circuit was designed and tested to measure the resonant frequency of micron-scale magnetoelastic (ME) biosensors using a pulsed wave excitation technique. In this circuit, a square pulse current is applied to an excitation coil to excite the vibration of ME biosensors and a pick-up coil is used to sense the ME biosensor's mechanical vibration and convert it to an electrical output signal. The output signal is filtered and amplified by a custom designed circuit to allow the measurement of the resonant frequency of the ME biosensor from which the detection of specific pathogens can be made. As a proof-in-concept experiment, JRB7 phage-coated ME biosensors were used to detect different concentrations of Bacillus anthracis Sterne strain spores. A statistically significant difference was observed for concentrations of 5 × 102 spore/ml and above.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores.

PubMed

Ghosh, Sourav K; Ostanin, Victor P; Johnson, Christian L; Lowe, Christopher R; Seshia, Ashwin A

2011-11-15

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R

PubMed Central

Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

2016-01-01

Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2−) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R. PMID:26927174

Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome▿ †

PubMed Central

Chitlaru, Theodor; Gat, Orit; Grosfeld, Haim; Inbar, Itzhak; Gozlan, Yael; Shafferman, Avigdor

2007-01-01

In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y. Gozlan, N. Ariel, and A. Shafferman, J. Bacteriol. 188:3551-3571, 2006). Using a battery of sera from B. anthracis-infected animals, in the present study we demonstrated that 49 of these proteins are immunogenic. Thirty-eight B. anthracis immunogens are documented in this study for the first time. The relative immunogenicities of the 49 secreted proteins appear to span a >10,000-fold range. The proteins eliciting the highest humoral response in the course of infection include, in addition to the well-established immunogens protective antigen (PA), Sap, and EA1, GroEL (BA0267), AhpC (BA0345), MntA (BA3189), HtrA (BA3660), 2,3-cyclic nucleotide diesterase (BA4346), collagen adhesin (BAS5205), an alanine amidase (BA0898), and an endopeptidase (BA1952), as well as three proteins having unknown functions (BA0796, BA0799, and BA0307). Of these 14 highly potent secreted immunogens, 11 are known to be associated with virulence and pathogenicity in B. anthracis or in other bacterial pathogens. Combining the results reported here with the results of a similar study of the membranal proteome of B. anthracis (T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, and E. Elhanany, Proteomics 4:677-691, 2004) and the results obtained in a functional genomic search for immunogens (O. Gat, H. Grosfeld, N. Ariel, I. Inbar, G. Zaide, Y. Broder, A. Zvi, T. Chitlaru, Z. Altboum, D. Stein, S. Cohen, and A. Shafferman, Infect. Immun. 74:3987-4001, 2006), we generated a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention. In a preliminary study, the efficacies of eight immunogens following DNA immunization of guinea pigs were compared to the efficacy of a PA DNA vaccine. All eight immunogens induced specific high antibody titers comparable to the titers elicited by PA; however, unlike PA, none of them provided protection against a lethal challenge (50 50% lethal doses) of virulent B. anthracis strain Vollum spores. PMID:17353282

A Mathematical Model of Anthrax Transmission in Animal Populations.

PubMed

Saad-Roy, C M; van den Driessche, P; Yakubu, Abdul-Aziz

2017-02-01

A general mathematical model of anthrax (caused by Bacillus anthracis) transmission is formulated that includes live animals, infected carcasses and spores in the environment. The basic reproduction number [Formula: see text] is calculated, and existence of a unique endemic equilibrium is established for [Formula: see text] above the threshold value 1. Using data from the literature, elasticity indices for [Formula: see text] and type reproduction numbers are computed to quantify anthrax control measures. Including only herbivorous animals, anthrax is eradicated if [Formula: see text]. For these animals, oscillatory solutions arising from Hopf bifurcations are numerically shown to exist for certain parameter values with [Formula: see text] and to have periodicity as observed from anthrax data. Including carnivores and assuming no disease-related death, anthrax again goes extinct below the threshold. Local stability of the endemic equilibrium is established above the threshold; thus, periodic solutions are not possible for these populations. It is shown numerically that oscillations in spore growth may drive oscillations in animal populations; however, the total number of infected animals remains about the same as with constant spore growth.

Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice

PubMed Central

Popova, Taissia G.; Espina, Virginia; Liotta, Lance A.; Popov, Serguei G.

2015-01-01

Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissesction. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and the protective capacity of the host. PMID:26091359

Spectrally-resolved fluorescence cross sections of aerosolized biological live agents and simulants using five excitation wavelengths in a BSL-3 laboratory.

PubMed

Pan, Yong-Le; Hill, Steven C; Santarpia, Joshua L; Brinkley, Kelly; Sickler, Todd; Coleman, Mark; Williamson, Chatt; Gurton, Kris; Felton, Melvin; Pinnick, Ronald G; Baker, Neal; Eshbaugh, Jonathan; Hahn, Jerry; Smith, Emily; Alvarez, Ben; Prugh, Amber; Gardner, Warren

2014-04-07

A system for measuring spectrally-resolved fluorescence cross sections of single bioaerosol particles has been developed and employed in a biological safety level 3 (BSL-3) facility at Edgewood Chemical and Biological Center (ECBC). It is used to aerosolize the slurry or solution of live agents and surrogates into dried micron-size particles, and to measure the fluorescence spectra and sizes of the particles one at a time. Spectrally-resolved fluorescence cross sections were measured for (1) bacterial spores: Bacillus anthracis Ames (BaA), B. atrophaeus var. globigii (BG) (formerly known as Bacillus globigii), B. thuringiensis israelensis (Bti), B. thuringiensis kurstaki (Btk), B. anthracis Sterne (BaS); (2) vegetative bacteria: Escherichia coli (E. coli), Pantoea agglomerans (Eh) (formerly known as Erwinia herbicola), Yersinia rohdei (Yr), Yersinia pestis CO92 (Yp); and (3) virus preparations: Venezuelan equine encephalitis TC83 (VEE) and the bacteriophage MS2. The excitation wavelengths were 266 nm, 273 nm, 280 nm, 365 nm and 405 nm.

Culturability of Bacillus spores on aerosol collection filters exposed to airborne combustion products of Al, Mg, and B·Ti.

PubMed

Adhikari, Atin; Yermakov, Michael; Indugula, Reshmi; Reponen, Tiina; Driks, Adam; Grinshpun, Sergey A

2016-05-01

Destruction of bioweapon facilities due to explosion or fire could aerosolize highly pathogenic microorganisms. The post-event air quality assessment is conducted through air sampling. A bioaerosol sample (often collected on a filter for further culture-based analysis) also contains combustion products, which may influence the microbial culturability and, thus, impact the outcome. We have examined the interaction between spores deposited on collection filters using two simulants of Bacillus anthracis [B. thuringiensis (Bt) and B. atrophaeus (referred to as BG)] and incoming combustion products of Al as well as Mg and B·Ti (common ingredient of metalized explosives). Spores extracted from Teflon, polycarbonate, mixed cellulose ester (MCE), and gelatin filters (most common filter media for bioaerosol sampling), which were exposed to combustion products during a short-term sampling, were analyzed by cultivation. Surprisingly, we observed that aluminum combustion products enhanced the culturability of Bt (but not BG) spores on Teflon filters increasing the culturable count by more than an order of magnitude. Testing polycarbonate and MCE filter materials also revealed a moderate increase of culturability although gelatin did not. No effect was observed with either of the two species interacting on either filter media with products originated by combustion of Mg and B·Ti. Sample contamination, spore agglomeration, effect of a filter material on the spore survival, changes in the spore wall ultrastructure and germination, as well as other factors were explored to interpret the findings. The study raises a question about the reliability of certain filter materials for collecting airborne bio-threat agents in combustion environments. Copyright © 2016 Elsevier Inc. All rights reserved.

Lethality of Bacillus Anthracis Spores Due to Short Duration Heating Measured Using Infrared Spectroscopy

DTIC Science & Technology

2005-03-01

images the light onto the detector at the focal plane (FP). 32 FP L5 HG L4 SH SL L3 NF L2 Figure 10. Spectrograph Stage of Raman Base Unit...the precise intensity at any given time. There was a small red light on the front of the instrument, as indicated in Figure 13, which would glow...Force Base , Ohio APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED. The views expressed in this thesis are those of the author and do not

A Cumulative Spore Killing Approach: Synergistic Sporicidal Activity of Dilute Peracetic Acid and Ethanol at Low pH Against Clostridium difficile and Bacillus subtilis Spores.

PubMed

Nerandzic, Michelle M; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J

2016-01-01

Background.  Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5-2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods.  We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results.  Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200-2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions.  Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin.

Evaluation of standardized sample collection, packaging, and ...

EPA Pesticide Factsheets

Journal Sample collection procedures and primary receptacle (sample container and bag) decontamination methods should prevent contaminant transfer between contaminated and non-contaminated surfaces and areas during bio-incident operations. Cross-contamination of personnel, equipment, or sample containers may result in the exfiltration of biological agent from the exclusion (hot) zone and have unintended negative consequences on response resources, activities and outcomes. The current study was designed to: (1) evaluate currently recommended sample collection and packaging procedures to identify procedural steps that may increase the likelihood of spore exfiltration or contaminant transfer; (2) evaluate the efficacy of currently recommended primary receptacle decontamination procedures; and (3) evaluate the efficacy of outer packaging decontamination methods. Wet- and dry-deposited fluorescent tracer powder was used in contaminant transfer tests to qualitatively evaluate the currently-recommended sample collection procedures. Bacillus atrophaeus spores, a surrogate for Bacillus anthracis, were used to evaluate the efficacy of spray- and wipe-based decontamination procedures.

Systematic evaluation of the efficacy of chlorine dioxide in decontamination of building interior surfaces contaminated with anthrax spores.

PubMed

Rastogi, Vipin K; Ryan, Shawn P; Wallace, Lalena; Smith, Lisa S; Shah, Saumil S; Martin, G Blair

2010-05-01

Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of Bacillus anthracis spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of Bacillus atrophaeus on stainless steel) was significantly less than that required for decontamination of most of the building materials tested.

Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

2012-03-01

B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

Development and field testing of a mobile chlorine dioxide generation system for the decontamination of buildings contaminated with Bacillus anthracis.

PubMed

Wood, Joseph P; Blair Martin, G

2009-05-30

The numerous buildings that became contaminated with Bacillus anthracis (the bacterium causing the disease anthrax) in 2001, and more recent B. anthracis - related events, point to the need to have effective decontamination technologies for buildings contaminated with biological threat agents. The U.S. Government developed a portable chlorine dioxide (ClO(2)) generation system to decontaminate buildings contaminated with B. anthracis spores, and this so-called mobile decontamination trailer (MDT) prototype was tested through a series of three field trials. The first test of the MDT was conducted at Fort McClellan in Anniston, AL. during October 2004. Four test attempts occurred over two weekends; however, a number of system problems resulted in termination of the activity prior to any ClO(2) introduction into the test building. After making several design enhancements and equipment changes, the MDT was subjected to a second test. During this test, extensive leak checks were made using argon and nitrogen in lieu of chlorine gas; each subsystem was checked for functionality, and the MDT was operated for 24h. This second test demonstrated the MDT flow and control systems functioned satisfactorily, and thus it was decided to proceed to a third, more challenging field trial. In the last field test, ClO(2) was generated and routed directly to the scrubber in a 12-h continuous run. Measurement of ClO(2) levels at the generator outlet showed that the desired production rate was not achieved. Additionally, only one of the two scrubbers performed adequately with regard to maintaining ClO(2) emissions below the limit. Numerous lessons were learned in the field trials of this ClO(2) decontamination technology.

The resistance of Bacillus atrophaeus spores to the bactericidal activity of peracetic acid is influenced by both the nature of the solid substrates and the mode of contamination.

PubMed

Grand, I; Bellon-Fontaine, M-N; Herry, J-M; Hilaire, D; Moriconi, F-X; Naïtali, M

2010-11-01

To evaluate the impact of the mode of contamination in relation with the nature of solid substrates on the resistance of spores of Bacillus atrophaeus -selected as surrogates of Bacillus anthracis- to a disinfectant, peracetic acid. Six materials confronted in urban and military environments were selected for their different structural and physicochemical properties. In parallel, two modes of contamination were examined, i.e. deposition and immersion. Deposition was used to simulate contamination by an aerosol and immersion by an extended contact with liquids. A pronounced difference in the biocontamination levels and spatial organization of spores was observed depending on the mode of contamination and the nature of the solid substrate considered, with consequences on decontamination. Contamination by immersion led to lower efficiency of peracetic acid decontamination than contamination by deposition. Infiltration of spores into porous materials after immersion is one reason. In contrast, the deposition mode aggregates cells at the surface of materials, explaining the similar disinfecting behaviour of porous and nonporous substrates when considering this inoculation route. The inoculation route was shown to be as influential a parameter as material characteristics (porosity and wettability) for decontamination efficacy. These results provide comparative information for the decontamination of B. atrophaeus spores in function of the mode of contamination and the nature of solid substrates. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology. No claim to French government works.

Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009.

PubMed

Wright, Jennifer Gordon; Quinn, Conrad P; Shadomy, Sean; Messonnier, Nancy

2010-07-23

These recommendations from the Advisory Committee on Immunization Practices (ACIP) update the previous recommendations for anthrax vaccine adsorbed (AVA) (CDC. Use of anthrax vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2000;49:1-20; CDC. Use of anthrax vaccine in response to terrorism: supplemental recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2002;51:1024-6) and reflect the status of anthrax vaccine supplies in the United States. This statement 1) provides updated information on anthrax epidemiology; 2) summarizes the evidence regarding the effectiveness and efficacy, immunogenicity, and safety of AVA; 3) provides recommendations for pre-event and preexposure use of AVA; and 4) provides recommendations for postexposure use of AVA. In certain instances, recommendations that did not change were clarified. No new licensed anthrax vaccines are presented. Substantial changes to these recommendations include the following: 1) reducing the number of doses required to complete the pre-event and preexposure primary series from 6 doses to 5 doses, 2) recommending intramuscular rather than subcutaneous AVA administration for preexposure use, 3) recommending AVA as a component of postexposure prophylaxis in pregnant women exposed to aerosolized Bacillus anthracis spores, 4) providing guidance regarding preexposure vaccination of emergency and other responder organizations under the direction of an occupational health program, and 5) recommending 60 days of antimicrobial prophylaxis in conjunction with 3 doses of AVA for optimal protection of previously unvaccinated persons after exposure to aerosolized B. anthracis spores.

Whole Genome Analysis of Injectional Anthrax Identifies Two Disease Clusters Spanning More Than 13 Years.

PubMed

Keim, Paul; Grunow, Roland; Vipond, Richard; Grass, Gregor; Hoffmaster, Alex; Birdsell, Dawn N; Klee, Silke R; Pullan, Steven; Antwerpen, Markus; Bayer, Brittany N; Latham, Jennie; Wiggins, Kristin; Hepp, Crystal; Pearson, Talima; Brooks, Tim; Sahl, Jason; Wagner, David M

2015-11-01

Anthrax is a rare disease in humans but elicits great public fear because of its past use as an agent of bioterrorism. Injectional anthrax has been occurring sporadically for more than ten years in heroin consumers across multiple European countries and this outbreak has been difficult to trace back to a source. We took a molecular epidemiological approach in understanding this disease outbreak, including whole genome sequencing of Bacillus anthracis isolates from the anthrax victims. We also screened two large strain repositories for closely related strains to provide context to the outbreak. Analyzing 60 Bacillus anthracis isolates associated with injectional anthrax cases and closely related reference strains, we identified 1071 Single Nucleotide Polymorphisms (SNPs). The synapomorphic SNPs (350) were used to reconstruct phylogenetic relationships, infer likely epidemiological sources and explore the dynamics of evolving pathogen populations. Injectional anthrax genomes separated into two tight clusters: one group was exclusively associated with the 2009-10 outbreak and located primarily in Scotland, whereas the second comprised more recent (2012-13) cases but also a single Norwegian case from 2000. Genome-based differentiation of injectional anthrax isolates argues for at least two separate disease events spanning > 12 years. The genomic similarity of the two clusters makes it likely that they are caused by separate contamination events originating from the same geographic region and perhaps the same site of drug manufacturing or processing. Pathogen diversity within single patients challenges assumptions concerning population dynamics of infecting B. anthracis and host defensive barriers for injectional anthrax. This work was supported by the United States Department of Homeland Security grant no. HSHQDC-10-C-00,139 and via a binational cooperative agreement between the United States Government and the Government of Germany. This work was supported by funds from the German Ministry of Defense (Sonderforschungsprojekt 25Z1-S-431,214). Support for sequencing was also obtained from Illumina, Inc. These sources had no role in the data generation or interpretation, and had not role in the manuscript preparation. We searched PubMed for any article published before Jun. 17, 2015, with the terms "Bacillus anthracis" and "heroin", or "injectional anthrax". Other than our previously published work (Price et al., 2012), we found no other relevant studies on elucidating the global phylogenetic relationships of B. anthracis strains associated with injectional anthrax caused by recreational heroin consumption of spore-contaminated drug. There were, however, publically available genome sequences of two strains involved (Price et al., 2012, Grunow et al., 2013) and the draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user (Ruckert et al., 2012) with only limited information on the genotyping of closely related strains (Price et al., 2012, Grunow et al., 2013). Injectional anthrax has been plaguing heroin drug users across Europe for more than 10 years. In order to better understand this outbreak, we assessed genomic relationships of all available injectional anthrax strains from four countries spanning a > 12 year period. Very few differences were identified using genome-based analysis, but these differentiated the isolates into two distinct clusters. This strongly supports a hypothesis of at least two separate anthrax spore contamination events perhaps during the drug production processes. Identification of two events would not have been possible from standard epidemiological analysis. These comprehensive data will be invaluable for classifying future injectional anthrax isolates and for future geographic attribution.

Systematic Evaluation of the Efficacy of Chlorine Dioxide in Decontamination of Building Interior Surfaces Contaminated with Anthrax Spores▿

PubMed Central

Rastogi, Vipin K.; Ryan, Shawn P.; Wallace, Lalena; Smith, Lisa S.; Shah, Saumil S.; Martin, G. Blair

2010-01-01

Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of Bacillus anthracis spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of Bacillus atrophaeus on stainless steel) was significantly less than that required for decontamination of most of the building materials tested. PMID:20305025

Anthrax: a continuing concern in the era of bioterrorism

PubMed Central

2005-01-01

Anthrax, a potentially fatal infection, is a virulent and highly contagious disease. It is caused by a gram-positive, toxigenic, spore-forming bacillus: Bacillus anthracis. For centuries, anthrax has caused disease in animals and, although uncommonly, in humans throughout the world. Descriptions of this naturally occurring disease begin in antiquity. Anthrax is primarily a disease of herbivores, which are infected by ingestion of spores from the soil. With the advent of modern microbiology, Pasteur developed the first successful anthrax vaccine in 1881. The incidence of the disease has continually decreased since the late 19th century, and animal vaccination programs drastically reduced the animal mortality from the disease. However, anthrax spores continue to be documented in soil samples from throughout the world. Research on anthrax as a biological weapon began more than 80 years ago, and today at least 17 nations are believed to have offensive biological weapons programs that include anthrax. Recent events in the USA have shown how society is affected by both hoax and real threats of anthrax bioweapons. This fourth article in the series on weapons of biowarfare/bioterrorism summarizes the historical background of anthrax as well as clinical and laboratory information useful for bioterrorism preparedness. PMID:16200179

Classification of select category A and B bacteria by Fourier transform infrared spectroscopy

NASA Astrophysics Data System (ADS)

Samuels, Alan C.; Snyder, A. Peter; St. Amant, Diane; Emge, Darren K.; Minter, Jennifer; Campbell, Mark; Tripathi, Ashish

2008-04-01

Relatively few reports have investigated the determination and classification of pathogens such as the National Institute of Allergy and Infectious Diseases (NIAID) Category A Bacillus anthracis spores and cells (BA), Yersinia species, Francisella tularensis (FT), and Category B Brucella species from FTIR spectra. We investigated the classification ability of the Fourier transform infrared (FTIR) spectra of viable pathogenic and non-pathogenic NIAID Category A and B bacteria. The impact of different growth media, growth time and temperature, rolling circle filter of the data, and wavelength range were investigated for their microorganism differentiation. Various 2-D PC plots provided differential degrees of separation with respect to the four viable, bacterial genera including the BA sub-categories of pathogenic spores, vegetative cells, and nonpathogenic vegetative cells. FT spectra were separated from that of the three other genera. The BA pathogenic spore strains 1029, LA1, and Ames were clearly differentiated from the rest of the dataset. Yersinia species were distinctly separated from the remaining dataset and could also be classified by growth media. This work provided evidence that FTIR spectroscopy can separate the four major pathogenic bacterial genera of NIAID Category A and B biological threat agents.

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis.

PubMed

Beuchat, Larry R; Pettigrew, Charles A; Tremblay, Mario E; Roselle, Brian J; Scouten, Alan J

2004-08-01

Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars

PubMed Central

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-01-01

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2–1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus. PMID:28298443

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

PubMed

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-05-05

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of vacuum filter sock surface sample collection method for Bacillus spores from porous and non-porous surfaces.

PubMed

Brown, Gary S; Betty, Rita G; Brockmann, John E; Lucero, Daniel A; Souza, Caroline A; Walsh, Kathryn S; Boucher, Raymond M; Tezak, Matthew S; Wilson, Mollye C

2007-07-01

Vacuum filter socks were evaluated for recovery efficiency of powdered Bacillus atrophaeus spores from two non-porous surfaces, stainless steel and painted wallboard and two porous surfaces, carpet and bare concrete. Two surface coupons were positioned side-by-side and seeded with aerosolized Bacillus atrophaeus spores. One of the surfaces, a stainless steel reference coupon, was sized to fit into a sample vial for direct spore removal, while the other surface, a sample surface coupon, was sized for a vacuum collection application. Deposited spore material was directly removed from the reference coupon surface and cultured for enumeration of colony forming units (CFU), while deposited spore material was collected from the sample coupon using the vacuum filter sock method, extracted by sonication and cultured for enumeration. Recovery efficiency, which is a measure of overall transfer effectiveness from the surface to culture, was calculated as the number of CFU enumerated from the filter sock sample per unit area relative to the number of CFU enumerated from the co-located reference coupon per unit area. The observed mean filter sock recovery efficiency from stainless steel was 0.29 (SD = 0.14, n = 36), from painted wallboard was 0.25 (SD = 0.15, n = 36), from carpet was 0.28 (SD = 0.13, n = 40) and from bare concrete was 0.19 (SD = 0.14, n = 44). Vacuum filter sock recovery quantitative limits of detection were estimated at 105 CFU m(-2) from stainless steel and carpet, 120 CFU m(-2) from painted wallboard and 160 CFU m(-2) from bare concrete. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling for biological agents such as Bacillus anthracis.

A Cumulative Spore Killing Approach: Synergistic Sporicidal Activity of Dilute Peracetic Acid and Ethanol at Low pH Against Clostridium difficile and Bacillus subtilis Spores

PubMed Central

Nerandzic, Michelle M.; Sankar C, Thriveen; Setlow, Peter; Donskey, Curtis J.

2016-01-01

Background. Alcohol-based hand sanitizers are the primary method of hand hygiene in healthcare settings, but they lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We previously demonstrated that acidification of ethanol induced rapid sporicidal activity, resulting in ethanol formulations with pH 1.5–2 that were as effective as soap and water washing in reducing levels of C difficile spores on hands. We hypothesized that the addition of dilute peracetic acid (PAA) to acidified ethanol would enhance sporicidal activity while allowing elevation of the pH to a level likely to be well tolerated on skin (ie, >3). Methods. We tested the efficacy of acidified ethanol solutions alone or in combination with PAA against C difficile and Bacillus subtilis spores in vitro and against nontoxigenic C difficile spores on hands of volunteers. Results. Acidification of ethanol induced rapid sporicidal activity against C difficile and to a lesser extent B subtilis. The addition of dilute PAA to acidified ethanol resulted in synergistic enhancement of sporicidal activity in a dose-dependent fashion in vitro. On hands, the addition of 1200–2000 ppm PAA enhanced the effectiveness of acidified ethanol formulations, resulting in formulations with pH >3 that were as effective as soap and water washing. Conclusions. Acidification and the addition of dilute PAA induced rapid sporicidal activity in ethanol. Our findings suggest that it may be feasible to develop effective sporicidal ethanol formulations that are safe and tolerable on skin. PMID:26885539

DNA capture elements for rapid detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

2004-08-01

DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

Laboratory-Scale Study in Determining the Decontamination Standards for Personnel Protective Equipment Used by Homeland Defense Personnel: Evaluation of Commercial Off-the-Shelf Technologies for Decontamination of Personnel Protective Equipment-Relevant Surfaces

DTIC Science & Technology

2008-06-01

Assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus Spores on Indoor Surfaces Using a Hydrogen Peroxide Gas...24-25% hydrogen peroxide (CAS # 7722-84-1), and 1-1.4% acetic acid. Clorox® bleach was diluted 1/ 1 0 th with sterile distilled water. Clean Earth...Peridox TM was diluted 1/6th with sterile distilled water. The disinfectants were used within 2 hr of their preparation. 2.2 Coupon Procurement Small size

Noninvasive Imaging Technologies Reveal Edema Toxin as a Key Virulence Factor in Anthrax

PubMed Central

Dumetz, Fabien; Jouvion, Grégory; Khun, Huot; Glomski, Ian Justin; Corre, Jean-Philippe; Rougeaux, Clémence; Tang, Wei-Jen; Mock, Michèle; Huerre, Michel; Goossens, Pierre Louis

2011-01-01

Powerful noninvasive imaging technologies enable real-time tracking of pathogen-host interactions in vivo, giving access to previously elusive events. We visualized the interactions between wild-type Bacillus anthracis and its host during a spore infection through bioluminescence imaging coupled with histology. We show that edema toxin plays a central role in virulence in guinea pigs and during inhalational infection in mice. Edema toxin (ET), but not lethal toxin (LT), markedly modified the patterns of bacterial dissemination leading, to apparent direct dissemination to the spleen and provoking apoptosis of lymphoid cells. Each toxin alone provoked particular histological lesions in the spleen. When ET and LT are produced together during infection, a specific temporal pattern of lesion developed, with early lesions typical of LT, followed at a later stage by lesions typical of ET. Our study provides new insights into the complex spatial and temporal effects of B. anthracis toxins in the infected host, suggesting a greater role than previously suspected for ET in anthrax and suggesting that therapeutic targeting of ET contributes to protection. PMID:21641378

Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

2004-12-01

Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

Towards a human oral vaccine for anthrax: the utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy.

PubMed

Baillie, Leslie W J; Rodriguez, Ana L; Moore, Stephen; Atkins, Helen S; Feng, Chiguang; Nataro, James P; Pasetti, Marcela F

2008-11-11

We previously demonstrated the ability of an orally administered attenuated Salmonella enterica serovar Typhimurium strain expressing the protective antigen (PA) of Bacillus anthracis to confer protection against lethal anthrax aerosol spore challenge [Stokes MG, Titball RW, Neeson BN, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75(April (4)):1827-34]. To extend the utility of this approach to humans we constructed variants of S. enterica serovar Typhi Ty21a, an attenuated typhoid vaccine strain licensed for human use, which expressed and exported PA via two distinct plasmid-based transport systems: the Escherichia coli HlyA haemolysin and the S. Typhi ClyA export apparatus. Murine immunogenicity studies confirmed the ability of these constructs, especially Ty21a expressing the ClyA-PA fusion protein, to stimulate strong PA-specific immune responses following intranasal immunization. These responses were further enhanced by a subsequent boost with either parenterally delivered recombinant PA or the licensed US human alum-adsorbed anthrax vaccine (AVA). Anthrax toxin neutralizing antibody responses using this prime-boost regimen were rapid, vigorous and broad in nature. The results of this study demonstrate the feasibility of employing a mucosal prime with a licensed Salmonella Typhi vaccine strain followed by a parenteral protein boost to stimulate rapid protective immunity against anthrax.

Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

PubMed

Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

2011-01-01

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Recombinant vaccine displaying the loop-neutralizing determinant from protective antigen completely protects rabbits from experimental inhalation anthrax.

PubMed

Oscherwitz, Jon; Yu, Fen; Jacobs, Jana L; Cease, Kemp B

2013-03-01

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2β2-2β3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD(50)) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines.

A synthetic peptide vaccine directed against the 2ß2-2ß3 loop of domain 2 of protective antigen protects rabbits from inhalation anthrax.

PubMed

Oscherwitz, Jon; Yu, Fen; Cease, Kemp B

2010-09-15

The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.

Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piepel, Gregory F.; Hutchison, Janine R.; Kaiser, Brooke L. D.

The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in × 2 in) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results are discussed in a separate report.« less

Recovery Efficiency, False Negative Rate, and Limit of Detection Performance of a Validated Macrofoam-Swab Sampling Method with Low Surface Concentrations of Two Bacillus anthracis Surrogates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piepel, Gregory F.; Hutchison, Janine R.; Deatherage Kaiser, Brooke L

The performance of a macrofoam-swab sampling method was evaluated using Bacillus anthracis Sterne (BAS) and Bacillus atrophaeus Nakamura (BG) spores applied at nine low target amounts (2-500 spores) to positive-control plates and test coupons (2 in. × 2 in.) of four surface materials (glass, stainless steel, vinyl tile, and plastic). Test results from cultured samples were used to evaluate the effects of surrogate, surface concentration, and surface material on recovery efficiency (RE), false negative rate (FNR), and limit of detection. For RE, surrogate and surface material had statistically significant effects, but concentration did not. Mean REs were the lowest formore » vinyl tile (50.8% with BAS, 40.2% with BG) and the highest for glass (92.8% with BAS, 71.4% with BG). FNR values ranged from 0 to 0.833 for BAS and 0 to 0.806 for BG, with values increasing as concentration decreased in the range tested (0.078 to 19.375 CFU/cm 2, where CFU denotes ‘colony forming units’). Surface material also had a statistically significant effect. A FNR-concentration curve was fit for each combination of surrogate and surface material. For both surrogates, the FNR curves tended to be the lowest for glass and highest for vinyl title. The FNR curves for BG tended to be higher than for BAS at lower concentrations, especially for glass. Results using a modified Rapid Viability-Polymerase Chain Reaction (mRV-PCR) analysis method were also obtained. The mRV-PCR results and comparisons to the culture results will be discussed in a subsequent report.« less

Towards Single-Shot Detection of Bacterial Endospores via Coherent Raman Spectroscopy

NASA Astrophysics Data System (ADS)

Pestov, Dmitry; Wang, Xi; Ariunbold, Gombojav; Murawski, Robert; Sautenkov, Vladimir; Sokolov, Alexei; Scully, Marlan

2007-10-01

Recent advances in coherent anti-Stokes Raman scattering (CARS) spectroscopy hold exciting promise to make the most out of now readily available ultrafast laser sources. Techniques have been devised to mitigate the nonresonant four-wave-mixing in favor of informative Raman-resonant signal. In particular, a hybrid technique for CARS (see Science 316, 265 (2007)) brings together the advantages of coherent broadband pump-Stokes excitation of molecular vibrations and their time-delayed but frequency-resolved probing via a spectrally narrowed and shaped laser pulse. We apply this technique to the problem of real-time detection of warfare bioagents and report single-shot acquisition of a distinct CARS spectrum from a small volume of B. subtilis endospores (˜10^4 spores), a harmless surrogate for B. anthracis. We study the dependence of the CARS signal on the energy of the ultrashort preparation pulses and find the limit on the pulse energy fluence (˜0.2 J/cm^2), imposed by the laser-induced damage of the spores.

Crystal Structures of the SpoIID Lytic Transglycosylases Essential for Bacterial Sporulation.

PubMed

Nocadello, Salvatore; Minasov, George; Shuvalova, Ludmilla S; Dubrovska, Ievgeniia; Sabini, Elisabetta; Anderson, Wayne F

2016-07-15

Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces▿

PubMed Central

Brown, Gary S.; Betty, Rita G.; Brockmann, John E.; Lucero, Daniel A.; Souza, Caroline A.; Walsh, Kathryn S.; Boucher, Raymond M.; Tezak, Mathew; Wilson, Mollye C.; Rudolph, Todd

2007-01-01

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of ±0.12, and for painted wallboard it was 0.29 with a standard deviation of ±0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of ±0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis. PMID:17122390

Evaluation of a wipe surface sample method for collection of Bacillus spores from nonporous surfaces.

PubMed

Brown, Gary S; Betty, Rita G; Brockmann, John E; Lucero, Daniel A; Souza, Caroline A; Walsh, Kathryn S; Boucher, Raymond M; Tezak, Mathew; Wilson, Mollye C; Rudolph, Todd

2007-02-01

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of +/-0.12, and for painted wallboard it was 0.29 with a standard deviation of +/-0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of +/-0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis.

Inhalation Anthrax: Dose Response and Risk Analysis

PubMed Central

Thran, Brandolyn; Morse, Stephen S.; Hugh-Jones, Martin; Massulik, Stacey

2008-01-01

The notion that inhalation of a single Bacillus anthracis spore is fatal has become entrenched nearly to the point of urban legend, in part because of incomplete articulation of the scientific basis for microbial risk assessment, particularly dose-response assessment. Risk analysis (ie, risk assessment, risk communication, risk management) necessitates transparency: distinguishing scientific facts, hypotheses, judgments, biases in interpretations, and potential misinformation. The difficulty in achieving transparency for biothreat risk is magnified by misinformation and poor characterization of both dose-response relationships and the driving mechanisms that cause susceptibility or resistance to disease progression. Regrettably, this entrenchment unnecessarily restricts preparedness planning to a single response scenario: decontaminate until no spores are detectable in air, water, or on surfaces—essentially forcing a zero-tolerance policy inconsistent with the biology of anthrax. We present evidence about inhalation anthrax dose-response relationships, including reports from multiple studies documenting exposures insufficient to cause inhalation anthrax in laboratory animals and humans. The emphasis of the article is clarification about what is known from objective scientific evidence for doses of anthrax spores associated with survival and mortality. From this knowledge base, we discuss the need for future applications of more formal risk analysis processes to guide development of alternative non-zero criteria or standards based on science to inform preparedness planning and other risk management activities. PMID:18582166

Recovery Efficiency and Limit of Detection of Aerosolized Bacillus anthracis Sterne from Environmental Surface Samples ▿

PubMed Central

Estill, Cheryl Fairfield; Baron, Paul A.; Beard, Jeremy K.; Hein, Misty J.; Larsen, Lloyd D.; Rose, Laura; Schaefer, Frank W.; Noble-Wang, Judith; Hodges, Lisa; Lindquist, H. D. Alan; Deye, Gregory J.; Arduino, Matthew J.

2009-01-01

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm2). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm2) or wipe or vacuum (929 cm2) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm2) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm2 for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm2 for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans. PMID:19429546

Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from environmental surface samples.

PubMed

Estill, Cheryl Fairfield; Baron, Paul A; Beard, Jeremy K; Hein, Misty J; Larsen, Lloyd D; Rose, Laura; Schaefer, Frank W; Noble-Wang, Judith; Hodges, Lisa; Lindquist, H D Alan; Deye, Gregory J; Arduino, Matthew J

2009-07-01

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm(2)). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm(2)) or wipe or vacuum (929 cm(2)) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm(2)) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm(2) for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm(2) for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.

THE SMALL ACID SOLUBLE PROTEINS (SASP α and SASP β) OF BACILLUS WEIHENSTEPHANENSIS AND B. MYCOIDES GROUP 2 ARE THE MOST DISTINCT AMONG THE B. CEREUS GROUP

PubMed Central

Callahan, Courtney; Fox, Karen; Fox, Alvin

2009-01-01

The Bacillus cereus group includes Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid-soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α-β type, was described here for the first time. PMID:19616612

Noninvasive imaging technologies reveal edema toxin as a key virulence factor in anthrax.

PubMed

Dumetz, Fabien; Jouvion, Grégory; Khun, Huot; Glomski, Ian Justin; Corre, Jean-Philippe; Rougeaux, Clémence; Tang, Wei-Jen; Mock, Michèle; Huerre, Michel; Goossens, Pierre Louis

2011-06-01

Powerful noninvasive imaging technologies enable real-time tracking of pathogen-host interactions in vivo, giving access to previously elusive events. We visualized the interactions between wild-type Bacillus anthracis and its host during a spore infection through bioluminescence imaging coupled with histology. We show that edema toxin plays a central role in virulence in guinea pigs and during inhalational infection in mice. Edema toxin (ET), but not lethal toxin (LT), markedly modified the patterns of bacterial dissemination leading, to apparent direct dissemination to the spleen and provoking apoptosis of lymphoid cells. Each toxin alone provoked particular histological lesions in the spleen. When ET and LT are produced together during infection, a specific temporal pattern of lesion developed, with early lesions typical of LT, followed at a later stage by lesions typical of ET. Our study provides new insights into the complex spatial and temporal effects of B. anthracis toxins in the infected host, suggesting a greater role than previously suspected for ET in anthrax and suggesting that therapeutic targeting of ET contributes to protection. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The efficacy and safety of nine South African medicinal plants in controlling Bacillus anthracis Sterne vaccine strain.

PubMed

Elisha, Ishaku Leo; Dzoyem, Jean-Paul; Botha, Francien S; Eloff, Jacobus Nicolaas

2016-01-08

Anthrax is a zoonotic disease caused by Bacillus anthracis, a Gram-positive spore-forming bacterium. The presence of the bacteria and the toxins in the blood of infected hosts trigger a cascade of pathological events leading to death. Nine medicinal plants with good activities against other bacteria were selected to determine their in vitro antibacterial activity against Bacillus anthracis Sterne strain. The cytotoxicity of the extracts on Vero kidney cells was also determined. The minimum inhibitory concentration (MIC) values of the extracts against Bacillus anthracis Sterne strain ranged from 0.02 to 0.31 mg/ml. Excellent MIC values were observed for the following plant species: Maesa lanceolata (0.02 mg/ml), Bolusanthus speciosus, Hypericum roeperianum, Morus mesozygia (0.04 mg/ml) and Pittosporum viridiflorum (0.08 mg/ml). The total antibacterial activity of the extracts ranged from 92 to 5562 ml/g. Total activity presents the volume to which the extract from 1 g of plant material can be diluted and still inhibit microbial growth. Maesa lanceolata and Hypericum roeperianum had the highest total activity with values of 5562 and 2999 ml/g respectively. The extracts of Calpurnia aurea had the lowest total activity (92 ml/g). The cytotoxicity determined on Vero cells indicated that most of the extracts were relatively non-toxic compared to doxorubicin (LC50 8.3 ± 1.76 μg/ml), except for the extracts of Maesa lanceolata, Elaeodendron croceum and Calpurnia aurea with LC50 values at 2.38 ± 0.25, 5.20 ± 0.24 and 13 ± 2.26 μg/ml respectively. The selectivity index (SI) ranged from 0.02 to 1.66. Hypericum roeperianum had the best selectivity index, (SI = 1.66) and Elaeodendron croceum had lowest value (SI = 0.02). The crude acetone extracts of the selected plant species had promising antibacterial activity against Bacillus anthracis. Maesa lanceolata extracts could be useful as a disinfectant and Hypericum roeperianum could be useful to protect animals based on its high total activity and selectivity index. Further investigation of these plant extracts may lead to the development of new therapeutic agents to protect humans or animals against anthrax.

Study of sporadical properties of crosslinked polyelectrolyte multilayers

NASA Astrophysics Data System (ADS)

Balu, Deebika

Polyelectrolyte multilayers (PEM) have become a highly studied class of materials due to the range of their applicability in many areas of research, including biology, chemistry and materials science. Recent advances in surface coatings have enabled modification of PEM surfaces to provide desirable properties such as controlled release, super-hydrophobicity, biocompatibility, antifouling and antibacterial properties. In the past decade, antimicrobial PEM coatings have been investigated as a safer alternative to the traditional disinfection methods that usually involve application of hazardous chemicals onto the surface to be cleaned. These antimicrobial coatings could be applied to common surfaces prone to colonization of bacteria (such as bench tops, faucet handles, etc) to supplement routine sanitization protocols by providing sustained antimicrobial activity. Vegetative bacteria (such as Escherichia coli) are more susceptible to antimicrobial agents than bacterial species that form spores. Hence, the antimicrobial activity of PEM coatings fabricated using Layer by Layer (LbL) technique were assayed using Bacillus anthracis spores (Sterne strain). In this thesis, the sporicidal effect of various polyelectrolyte multilayer coatings containing cross-linked polymers immersed in bleach have been evaluated as potential augmentation to existing disinfection methods.

Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters

PubMed Central

Calfee, M. Worth; Rose, Laura J.; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal

2016-01-01

The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37 mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p ≤ 0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p > 0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident. PMID:24184312

Evaluation of sampling methods for Bacillus spore-contaminated HVAC filters.

PubMed

Calfee, M Worth; Rose, Laura J; Tufts, Jenia; Morse, Stephen; Clayton, Matt; Touati, Abderrahmane; Griffin-Gatchalian, Nicole; Slone, Christina; McSweeney, Neal

2014-01-01

The objective of this study was to compare an extraction-based sampling method to two vacuum-based sampling methods (vacuum sock and 37mm cassette filter) with regards to their ability to recover Bacillus atrophaeus spores (surrogate for Bacillus anthracis) from pleated heating, ventilation, and air conditioning (HVAC) filters that are typically found in commercial and residential buildings. Electrostatic and mechanical HVAC filters were tested, both without and after loading with dust to 50% of their total holding capacity. The results were analyzed by one-way ANOVA across material types, presence or absence of dust, and sampling device. The extraction method gave higher relative recoveries than the two vacuum methods evaluated (p≤0.001). On average, recoveries obtained by the vacuum methods were about 30% of those achieved by the extraction method. Relative recoveries between the two vacuum methods were not significantly different (p>0.05). Although extraction methods yielded higher recoveries than vacuum methods, either HVAC filter sampling approach may provide a rapid and inexpensive mechanism for understanding the extent of contamination following a wide-area biological release incident. Published by Elsevier B.V.

Investigation of Anthrax Cases in North-East China, 2010-2014.

PubMed

Zhou, Wei; Sun, Yang; Zhu, Lingwei; Zhou, Bo; Liu, Jun; Ji, Xue; Wang, Xiaofeng; Wang, Nan; Gu, Guibo; Feng, Shuzhang; Qian, Jun; Guo, Xuejun

2015-01-01

We determined the genotypes of seven Bacillus anthracis strains that were recovered from nine anthrax outbreaks in North-East China from 2010 to 2014, and two approved vaccine strains that are currently in use in China. The causes of these cases were partly due to local farmers being unaware of the presence of anthrax, and butchers with open wounds having direct contact with anthrax-contaminated meat products. The genotype of five of the seven recovered strains was A.Br.001/002 sub-lineage, which was concordant with previously published research. The remaining two cases belongs to the A.Br.Ames sub-lineage. Both of these strains displayed an identical SNR pattern, which was the first time that this genotype was identified in North-East China. Strengthening education in remote villages of rural China is an important activity aimed at fostering attempts to prevent and control anthrax. The genotype of the vaccine strain Anthrax Spore Vaccine No.II was A.Br.008/009 and A.Br.001/002 for the vaccine strain Anthrax Spore Vaccine Non-capsulated. Further studies of their characteristics are clearly warranted.

The State Laboratory of Hygiene's role in terrorism preparedness and response.

PubMed

Hintzman, Peggy L

2003-01-01

In the fall of 2001, the national public health system found itself responding to acts of terrorism. The intentional release of Bacillus anthracis spores on the East Coast tested the capacity of all state public health laboratories to respond. The impact on the public health system extended to the Wisconsin State Laboratory of Hygiene (WSLH). Fortunately, participation in the National Laboratory Response Network helped the WSLH meet the challenge of 24 hour/7 days a week coverage, and subsequent federal funding increases have enabled the WSLH to expand its technical capabilities and provide training and outreach to other Wisconsin laboratories to prepare them for their roles in man-made or naturally-occurring public health emergencies.

Fifth international fungus spore conference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timberlake, W.E.

1993-04-01

This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.

Selection and optimization of Bacillus atrophaeus inoculum medium and its effect on spore yield and thermal resistance.

PubMed

Sella, Sandra Regina B R; Dlugokenski, Regina Elizabete F; Guizelini, Belquis P; Vandenberghe, Luciana P S; Medeiros, Adriane B P; Pandey, Ashok; Soccol, Carlos Ricardo

2008-12-01

Bacillus atrophaeus's spores are used as biological indicators to monitor sterilization processes and as a Bacillus anthracis surrogate in the development and validation of biosafety methods. The regular use of biological indicators to evaluate the efficiency of sterilization processes is a legal requirement for health services. However, its high cost hinders its widespread use. Aiming at developing a cost-effective inoculum medium, soybean molasses and nutrient-supplemented vinasse were evaluated for their effectiveness in solid-state fermentation (SSF). In biomass production, the results demonstrated that all tested compositions favor growth by providing the nutritional demands of the microorganism. Optimum casein peptone and soybean molasses concentration (1.0%, 2.5%, or 4.0%) was determined by a 2((2-0)) factorial experimental design. The results have showed a positive influence of peptone on biomass production. In order to define peptone final concentration (4.0% or 6.0%), a 2(2) factorial experimental design was used. An optimized medium containing 4.0% soybean molasses and 4.0% casein peptone was similar in performance to a synthetic control medium (tryptone soy broth) in dry-heat thermal-resistant spore production by SSF. An experiment performed under optimum SSF conditions resulted in 1.9 x 10(10) CFU g(-1) dry matter with D (160 degrees C) = 5.2 +/- 0.2 min.

Fifth international fungus spore conference. [Abstracts]: Final technical report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timberlake, W.E.

1993-04-01

This folio contains the proceedings of the Fifth International Fungal Spore Conference held August 17-21, 1991 at the Unicoi State Park at Helen, Georgia. The volume contains abstracts of each oral presentation as well as a collection of abstracts describing the poster sessions. Presentations were organized around the themes (1) Induction of Sporulation, (2) Nuclear Division, (3) Spore Formation, (4) Spore Release and Dispersal, and (4) Spore Germination.

Detection of Biological Pathogens Using Multiple Wireless Magnetoelastic Biosensors

NASA Astrophysics Data System (ADS)

Shen, Wen

A number of recent, high-profile incidences of food-borne illness spreading through the food supply and the use of anthrax by terrorists after the September 11, 2001 attacks have demonstrated the need for new technologies that can rapidly detect the presence of biological pathogens. A bevy of biosensors show excellent detection sensitivity and specificity. However, false positive and false negative signals remain one of the primary reasons that many of these newly developed biosensors have not found application in the marketplace. The research described in this dissertation focuses on developing a free-standing magnetoelastic based bio-sensing system using a pulse method. This method allows fast detection, eliminates the bias magnetic field that is necessary in current methods, makes the system more simply and suitable for in-field detection. This system has two pairs of transformer coils, where a measurement sensor and a control sensor can be put in each pair of coils. The control sensor is used to compensate for environmental variables. The effect of pulse power on the performance of the magnetoelastic sensors in the pulse system is studied. The system is found to have excellent stability, good detection repeatability when used with multiple sensors. This research has investigated and demonstrated a multiple sensors approach. Because it will involve the simultaneous measurement of many sensors, it will significantly reduce problems encountered with false positive indications. The positioning and interference of sensors are investigated. By adding a multi-channel structure to the pulse detection system, the effect of sensor interference is minimized. The result of the repeatability test shows that the standard deviation when measuring three 1 mm magnetoelastic sensors is around 500 Hz, which is smaller than the minimum requirement for actual spores/bacteria detection. Magnetoelastic sensors immobilized with JRB7 phages and E2 phages have been used to specifically detect Bacillus anthracis spores and Salmonella typhimurium bacteria. The real-time monitoring of the detection of B. anthracis spores in a flowing system was performed using 2 mm sensors and 1 mm sensors. The detection of S. typhimurium in air has been performed using the pulse based system with both single and grouped sensors. Because grouped sensor detection involves the simultaneous measurement of many sensors, statistical evaluation shows that it can significantly reduce problems encountered with false positive indications. This method has been implemented in an investigation of a method that allows direct detection of S. typhimurium on cantaloupe surfaces. It has been demonstrated that multiple E2 phage based magnetoelastic sensors are able to detect Salmonella directly on fresh cantaloupe surfaces. Confirmation of the spore or bacteria binding to the sensor surfaces was achieved through SEM study of the sensor surfaces.

Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.

PubMed

Barth, Holger; Aktories, Klaus; Popoff, Michel R; Stiles, Bradley G

2004-09-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

PubMed Central

Barth, Holger; Aktories, Klaus; Popoff, Michel R.; Stiles, Bradley G.

2004-01-01

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria. PMID:15353562

[Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice].

PubMed

Wang, Xiaolin; Chi, Xiangyang; Liu, Ju; Liu, Weicen; Liu, Shuling; Qiu, Shunfang; Wen, Zhonghua; Fan, Pengfei; Liu, Kun; Song, Xiaohong; Fu, Ling; Zhang, Jun; Yu, Changming

2016-11-25

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

Protection against anthrax and plague by a combined vaccine in mice and rabbits.

PubMed

Ren, Jun; Dong, Dayong; Zhang, Jinlong; Zhang, Jun; Liu, Shuling; Li, Bing; Fu, Ling; Xu, Junjie; Yu, Changming; Hou, Lihua; Li, Jianmin; Chen, Wei

2009-12-09

The protective antigen (PA) of Bacillus anthracis and the Fraction 1 Capsular Antigen (F1 antigen), V antigen of Yersinia pestis have been demonstrated to be potential immunogens and candidate vaccine sub-units against anthrax and plague respectively. In this study, the authors have investigated the antibody responses and the protective efficacy when the antigens were administered separately or in combination intramuscularly formulation adsorbed to an aluminum hydroxide adjuvant. Results show that immunized rF1 + rV and rPA antigen together was as effective as separately for induction of serological antibody response, and these titers were maintained for over 1 year in mice. An isotype analysis of the serum indicates that the co-administration of these antigens did not influence the antigen-specific IgG1/IgG2a ratio which was consistent with a Th2 bias. Furthermore, the combined vaccine comprising the protein antigens rF1 + rV + rPA has been demonstrated to protect mice from subcutaneous challenge with 10(7) colony-forming units (CFU) virulent Y. pestis strain, and to fully protect rabbit against subcutaneous challenge with 1.2x10(5) colony-forming units (CFU) virulent B. anthracis spores. These data show that the protective efficacy was unaffected when the antigens were administered in combination.

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier.

PubMed

Schmid, Stefan; Seiler, Cornelia; Gerecke, Andreas C; Hächler, Herbert; Hilbi, Hubert; Frey, Joachim; Weidmann, Simon; Meier, Lukas; Berchtold, Christian; Zenobi, Renato

2013-07-15

Degradation of non-volatile organic compounds-environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)-in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0m s(-1) (3200Lmin(-1)), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10Lmin(-1). Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10Hz or 50Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative "degradation" efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials.

PubMed

Wood, J P; Calfee, M W; Clayton, M; Griffin-Gatchalian, N; Touati, A

2011-12-01

We evaluated whether lowering pH (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying pH and FAC levels affected bleach stability. Acidified bleach solutions with pH levels of 4.5, 6 and 7.5 and FAC levels between 5000 and 10,000 ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the pH or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at pH of 7.5 was significantly less effective than bleach at a pH of 4.5 or 6. The FAC levels in the bleach were the most stable at pH 4.5, and stability at pH 4.5 was not significantly affected by the initial FAC level. It may be advisable to use bleach solutions with lower pH (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

INTER-ALPHA INHIBITOR PROTEINS: A NOVEL THERAPEUTIC STRATEGY FOR EXPERIMENTAL ANTHRAX INFECTION

PubMed Central

Opal, Steven M.; Lim, Yow-Pin; Cristofaro, Patricia; Artenstein, Andrew W.; Kessimian, Noubar; DelSesto, David; Parejo, Nicolas; Palardy, John E.; Siryaporn, Edward

2010-01-01

Human inter-alpha-inhibitor proteins (IaIp) are endogenous human plasma proteins that function as serine protease inhibitors. IaIp can block the systemic release of proteases in sepsis and block furin-mediated assembly of protective antigen, an essential stop in the intracellular delivery of the anthrax exotoxins, lethal toxin and edema toxin. IaIp administered on hour or up to 24 hours after spore challenge with Bacillus anthracis Sterne strain protected mice from lethality if administered with antimicrobial therapy (p<.001). These human plasma proteins possess combined actions against anthrax as general inhibitors of excess serine proteases in sepsis and specific inhibitors of anthrax toxin assembly. IaIp could represent a novel adjuvant therapy for the treatment of established anthrax infection. PMID:20523269

Single-shot detection of bacterial endospores via coherent Raman spectroscopy.

PubMed

Pestov, Dmitry; Wang, Xi; Ariunbold, Gombojav O; Murawski, Robert K; Sautenkov, Vladimir A; Dogariu, Arthur; Sokolov, Alexei V; Scully, Marlan O

2008-01-15

Recent advances in coherent Raman spectroscopy hold exciting promise for many potential applications. For example, a technique, mitigating the nonresonant four-wave-mixing noise while maximizing the Raman-resonant signal, has been developed and applied to the problem of real-time detection of bacterial endospores. After a brief review of the technique essentials, we show how extensions of our earlier experimental work [Pestov D, et al. (2007) Science 316:265-268] yield single-shot identification of a small sample of Bacillus subtilis endospores (approximately 10(4) spores). The results convey the utility of the technique and its potential for "on-the-fly" detection of biohazards, such as Bacillus anthracis. The application of optimized coherent anti-Stokes Raman scattering scheme to problems requiring chemical specificity and short signal acquisition times is demonstrated.

Comparative performance of a licensed anthrax vaccine versus electroporation based delivery of a PA encoding DNA vaccine in rhesus macaques.

PubMed

Livingston, Brian D; Little, Stephen F; Luxembourg, Alain; Ellefsen, Barry; Hannaman, Drew

2010-01-22

DNA vaccination is a promising immunization strategy that could be applied in the development of vaccines for a variety of prophylactic and therapeutic indications. Utilizing anthrax protective antigen as a model antigen, we demonstrate that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection. Two administrations of a DNA vaccine with electroporation elicited anthrax toxin neutralizing antibody responses in 100% of rhesus macaques. Toxin neutralizing antibodies were sustained for the nearly 1-year study duration and were correlated with protection against subsequent lethal Bacillus anthracis spore challenge. Collectively, electroporation mediated DNA vaccination conferred protection comparable to that observed following vaccination with an FDA approved anthrax vaccine.

Vaccines and bioterrorism: smallpox and anthrax.

PubMed

Kimmel, Sanford R; Mahoney, Martin C; Zimmerman, Richard K

2003-01-01

Because of the success of vaccination and the ring strategy in eradicating smallpox from the world, smallpox vaccine has not been recommended for the United States civilian populations for decades. Given the low but possible threat of bioterrorism, smallpox vaccination is now recommended for those teams investigating potential smallpox cases and for selected personnel of acute-care hospitals who would be needed to care for victims in the event of a terrorist attack. Treatment and post-exposure prophylaxis for anthrax are ciprofloxacin or doxycycline. Anthrax vaccine alone is not effective for post-exposure prevention of anthrax; vaccination is accompanied by 60 days of antibiotic therapy. In addition to military use, anthrax vaccine is recommended for pre-exposure use in those persons whose work involves repeated exposure to Bacillus anthracis spores.

Considerations for estimating microbial environmental data concentrations collected from a field setting

PubMed Central

Silvestri, Erin E; Yund, Cynthia; Taft, Sarah; Bowling, Charlena Yoder; Chappie, Daniel; Garrahan, Kevin; Brady-Roberts, Eletha; Stone, Harry; Nichols, Tonya L

2017-01-01

In the event of an indoor release of an environmentally persistent microbial pathogen such as Bacillus anthracis, the potential for human exposure will be considered when remedial decisions are made. Microbial site characterization and clearance sampling data collected in the field might be used to estimate exposure. However, there are many challenges associated with estimating environmental concentrations of B. anthracis or other spore-forming organisms after such an event before being able to estimate exposure. These challenges include: (1) collecting environmental field samples that are adequate for the intended purpose, (2) conducting laboratory analyses and selecting the reporting format needed for the laboratory data, and (3) analyzing and interpreting the data using appropriate statistical techniques. This paper summarizes some key challenges faced in collecting, analyzing, and interpreting microbial field data from a contaminated site. Although the paper was written with considerations for B. anthracis contamination, it may also be applicable to other bacterial agents. It explores the implications and limitations of using field data for determining environmental concentrations both before and after decontamination. Several findings were of interest. First, to date, the only validated surface/sampling device combinations are swabs and sponge-sticks on stainless steel surfaces, thus limiting availability of quantitative analytical results which could be used for statistical analysis. Second, agreement needs to be reached with the analytical laboratory on the definition of the countable range and on reporting of data below the limit of quantitation. Finally, the distribution of the microbial field data and statistical methods needed for a particular data set could vary depending on these data that were collected, and guidance is needed on appropriate statistical software for handling microbial data. Further, research is needed to develop better methods to estimate human exposure from pathogens using environmental data collected from a field setting. PMID:26883476

Multispectral analysis of biological agents to implement a quick tool for stand-off biological detection

NASA Astrophysics Data System (ADS)

Carestia, M.; Pizzoferrato, R.; Lungaroni, M.; Gabriele, J.; Ludovici, G. M.; Cenciarelli, O.; Gelfusa, M.; Murari, A.; Malizia, A.; Gaudio, P.

2015-10-01

With the aim of identifying an approach to exploit the differences in the fluorescence signatures of biological agents BAs, we have investigated the response of some BAs simulants to a set of different excitation wavelengths in the UV spectral range (i.e. 266, 273, 280, 300, 340, 355 nm). Our preliminary results on bacterial spores and vegetative forms, dispersed in water, showed that the differences in the fluorescence spectra can be enhanced, and more easily revealed, by using different excitation wavelengths. Specifically, the photo luminescence (PL) spectra coming from different species of Bacillus, in the form of spores (used as simulants of Bacillus anthracis), show significant differences under excitation at all the wavelengths, with slightly larger differences at 300, 340, 355 nm. On the other hand, the vegetative forms of two Bacillus species, did not show any appreciable difference, i.e. the PL spectra are virtually identical, for the excitation wavelengths of 266, 273, 280 nm. Conversely, small yet appreciable difference appear at 300, 340, 355 nm. Finally, large difference appear between the spore and the vegetative form of each species at all the wavelengths, with slightly larger variations at 300, 340, 355 nm. Together, these preliminary results support the hypothesis that a multi-wavelength approach could be used to improve the sensitivity and specificity of UV-LIF based BAs detection systems. The second step of this work concerns the application of a Support Vector Regression (SVR) method, as evaluated in our previous work to define a methodology for the setup of a multispectral database for the stand-off detection of BAs.

A case report of inhalation anthrax acquired naturally.

PubMed

Azarkar, Zohreh; Bidaki, Majid Zare

2016-03-03

Anthrax is a zoonotic occupational disease caused by Bacillus anthracis, a rod-shaped immobile aerobic gram-positive bacteria with spore. Anthrax occurs in humans randomly and with low frequency. Most cases of anthrax are acquired through contact with infected animals or contaminated animal products. This old disease became particularly important since 2001 that the biological spores were exploited in America. Depending on the transmission method of the disease, clinical manifestations occur in three classes: Cutaneous, respiratory, and gastrointestinal anthrax. The respiratory form is considered as the most fatal and a rare form of anthrax intending to show complicated and unusual manifestations. In this case report a rare case of inhalation anthrax acquired naturally in southeast of Iran is presented. A blind 65-year-old man, living in a rural area, was admitted with respiratory infection, fever, dyspnea, loss of appetite, and myalgia. The patient was treated with outpatient antibiotics a week ago. After admission, the patient was again treated for pneumonia, but there was no improvement despite treatment and the patient was suffering from septicemia symptoms. Radiographic images showed wide mediastinum. Bacillus anthracis was isolated from blood and sputum culture and the results were confirmed by colony morphology, biochemical reactions and PCR. The treatment was changed to ciprofloxacin, clindamycin, and penicillin. On the second day of anthrax treatment, the patient was complicated with jaundice, elevation of liver enzymes, and a significant drop in hemoglobin, hematocrit, and platelet despite lack of obvious bleeding and was complicated with respiratory distress and sepsis and died a week after treatment. We could discover no specific exposure associated with anthrax infection for this patient. However, due to being located in an endemic and enzootic area, it is proposed that the exposure occurred through contact with infected airborne dust or an unknown contaminated item. Despite many advances in preventing anthrax, still some rare cases of respiratory and complicated anthrax are emerging. With regard to the threat of bioterrorism, medical staff's sensitivity to the clinical syndrome, methods of prophylaxis and treatment of anthrax must be raised. Fast diagnosis and successful treatment the lethal cases of this infection are of utmost important.

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores.

PubMed

Stachowiak, Jeanne C; Shugard, Erin E; Mosier, Bruce P; Renzi, Ronald F; Caton, Pamela F; Ferko, Scott M; Van de Vreugde, James L; Yee, Daniel D; Haroldsen, Brent L; VanderNoot, Victoria A

2007-08-01

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.

Recombinant protective antigen 102 (rPA102): profile of a second-generation anthrax vaccine.

PubMed

Keitel, Wendy A

2006-08-01

Recent terrorist attacks involving the use of Bacillus anthracis spores have stimulated interest in the development of new vaccines for anthrax prevention. Studies of the pathogenesis of anthrax and of the immune responses following infection and immunization underscore the pivotal role that antibodies to the protective antigen play in protection. The most promising vaccine candidates contain purified recombinant protective antigen. Clinical trials of one of these, recombinant protective antigen (rPA)102, are underway. Initial results suggest that rPA102 is well tolerated and immunogenic. Additional trials are necessary to identify optimal formulations and immunization regimens for pre- and postexposure prophylaxis. Future licensure of these and other candidate vaccines will depend on their safety and immunogenicity profiles in humans, and their ability to confer protection in animal models of inhalational anthrax.

Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.

PubMed

Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

2012-07-06

Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined. Published by Elsevier Ltd.

ANTHRAX IN THE MACKENZIE WOOD BISON (BISON BISON ATHABASCAE) POPULATION: 2012 ANTHRAX OUTBREAK AND HISTORICAL EXPOSURE IN NONOUTBREAK YEARS.

PubMed

New, Dallas; Elkin, Brett; Armstrong, Terry; Epp, Tasha

2017-10-01

Anthrax, caused by the spore-forming bacterium Bacillus anthracis, poses a threat to wood bison (Bison bison athabascae) conservation. We used descriptive epidemiology to characterize a large outbreak of anthrax in the Mackenzie bison population in the Northwest Territories, Canada, in 2012 and investigated historical serologic exposure of the bison to the bacterium in nonoutbreak years. Between late June and early August 2012, 451 bison carcasses were detected; mortality peaked from 13-19 July. A substantial number of calves, yearlings, and adult females died in the 2012 outbreak, unlike in two previous anthrax outbreaks in this population that killed mostly mature males. On the basis of the difference in estimates of population size prior to the outbreak (2012) and after the outbreak (2013), it is possible that not all dead bison were found during the outbreak. We assessed serologic history of exposure to B. anthracis by using samples from the Mackenzie wood bison population collected between 1986 and 2009. Overall, 87 of 278 samples were positive (31%). Seroprevalence was lower in females (18%, 10/55) than males (36%, 72/203). The highest proportion of positive submissions (90%) was from 1994, the year following the only anthrax outbreak within the historical data set. Both adult males and females had a higher likelihood of being seropositive than the younger age categories. There was a trend toward declining antibody levels between the 1993 and 2012 outbreak years.

Destruction of Spores on Building Decontamination Residue in a Commercial Autoclave▿

PubMed Central

Lemieux, P.; Sieber, R.; Osborne, A.; Woodard, A.

2006-01-01

The U.S. Environmental Protection Agency conducted an experiment to evaluate the effectiveness of a commercial autoclave for treating simulated building decontamination residue (BDR). The BDR was intended to simulate porous materials removed from a building deliberately contaminated with biological agents such as Bacillus anthracis (anthrax) in a terrorist attack. The purpose of the tests was to assess whether the standard operating procedure for a commercial autoclave provided sufficiently robust conditions to adequately destroy bacterial spores bound to the BDR. In this study we investigated the effects of several variables related to autoclaving BDR, including time, temperature, pressure, item type, moisture content, packing density, packing orientation, autoclave bag integrity, and autoclave process sequence. The test team created simulated BDR from wallboard, ceiling tiles, carpet, and upholstered furniture, and embedded in the BDR were Geobacillus stearothermophilus biological indicator (BI) strips containing 106 spores and thermocouples to obtain time and temperature profile data associated with each BI strip. The results indicated that a single standard autoclave cycle did not effectively decontaminate the BDR. Autoclave cycles consisting of 120 min at 31.5 lb/in2 and 275°F and 75 min at 45 lb/in2 and 292°F effectively decontaminated the BDR material. Two sequential standard autoclave cycles consisting of 40 min at 31.5 lb/in2 and 275°F proved to be particularly effective, probably because the second cycle's evacuation step pulled the condensed water out of the pores of the materials, allowing better steam penetration. The results also indicated that the packing density and material type of the BDR in the autoclave could have a significant impact on the effectiveness of the decontamination process. PMID:17012597

Reaerosolization of Spores from Flooring Surfaces To Assess the Risk of Dissemination and Transmission of Infections.

PubMed

Paton, Susan; Thompson, Katy-Anne; Parks, Simon R; Bennett, Allan M

2015-08-01

The aim of this study was to quantify reaerosolization of microorganisms caused by walking on contaminated flooring to assess the risk to individuals accessing areas contaminated with pathogenic organisms, for example, spores of Bacillus anthracis. Industrial carpet and polyvinyl chloride (PVC) floor coverings were contaminated with aerosolized spores of Bacillus atrophaeus by using an artist airbrush to produce deposition of ∼10(3) to 10(4) CFU · cm(-2). Microbiological air samplers were used to quantify the particle size distribution of the aerosol generated when a person walked over the floorings in an environmental chamber. Results were expressed as reaerosolization factors (percent per square centimeter per liter), to represent the ratio of air concentration to surface concentration generated. Walking on carpet generated a statistically significantly higher reaerosolization factor value than did walking on PVC (t = 20.42; P < 0.001). Heavier walking produced a statistically significantly higher reaerosolization factor value than did lighter walking (t = 12.421; P < 0.001). Height also had a statistically significant effect on the reaerosolization factor, with higher rates of recovery of B. atrophaeus at lower levels, demonstrating a height-dependent gradient of particle reaerosolization. Particles in the respirable size range were recovered in all sampling scenarios (mass mean diameters ranged from 2.6 to 4.1 μm). The results of this study can be used to produce a risk assessment of the potential aerosol exposure of a person accessing areas with contaminated flooring in order to inform the choice of appropriate respiratory protective equipment and may aid in the selection of the most suitable flooring types for use in health care environments, to reduce aerosol transmission in the event of contamination. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Reaerosolization of Spores from Flooring Surfaces To Assess the Risk of Dissemination and Transmission of Infections

PubMed Central

Thompson, Katy-Anne; Parks, Simon R.; Bennett, Allan M.

2015-01-01

The aim of this study was to quantify reaerosolization of microorganisms caused by walking on contaminated flooring to assess the risk to individuals accessing areas contaminated with pathogenic organisms, for example, spores of Bacillus anthracis. Industrial carpet and polyvinyl chloride (PVC) floor coverings were contaminated with aerosolized spores of Bacillus atrophaeus by using an artist airbrush to produce deposition of ∼103 to 104 CFU · cm−2. Microbiological air samplers were used to quantify the particle size distribution of the aerosol generated when a person walked over the floorings in an environmental chamber. Results were expressed as reaerosolization factors (percent per square centimeter per liter), to represent the ratio of air concentration to surface concentration generated. Walking on carpet generated a statistically significantly higher reaerosolization factor value than did walking on PVC (t = 20.42; P < 0.001). Heavier walking produced a statistically significantly higher reaerosolization factor value than did lighter walking (t = 12.421; P < 0.001). Height also had a statistically significant effect on the reaerosolization factor, with higher rates of recovery of B. atrophaeus at lower levels, demonstrating a height-dependent gradient of particle reaerosolization. Particles in the respirable size range were recovered in all sampling scenarios (mass mean diameters ranged from 2.6 to 4.1 μm). The results of this study can be used to produce a risk assessment of the potential aerosol exposure of a person accessing areas with contaminated flooring in order to inform the choice of appropriate respiratory protective equipment and may aid in the selection of the most suitable flooring types for use in health care environments, to reduce aerosol transmission in the event of contamination. PMID:25979883

Destruction of spores on building decontamination residue in a commercial autoclave.

PubMed

Lemieux, P; Sieber, R; Osborne, A; Woodard, A

2006-12-01

The U.S. Environmental Protection Agency conducted an experiment to evaluate the effectiveness of a commercial autoclave for treating simulated building decontamination residue (BDR). The BDR was intended to simulate porous materials removed from a building deliberately contaminated with biological agents such as Bacillus anthracis (anthrax) in a terrorist attack. The purpose of the tests was to assess whether the standard operating procedure for a commercial autoclave provided sufficiently robust conditions to adequately destroy bacterial spores bound to the BDR. In this study we investigated the effects of several variables related to autoclaving BDR, including time, temperature, pressure, item type, moisture content, packing density, packing orientation, autoclave bag integrity, and autoclave process sequence. The test team created simulated BDR from wallboard, ceiling tiles, carpet, and upholstered furniture, and embedded in the BDR were Geobacillus stearothermophilus biological indicator (BI) strips containing 10(6) spores and thermocouples to obtain time and temperature profile data associated with each BI strip. The results indicated that a single standard autoclave cycle did not effectively decontaminate the BDR. Autoclave cycles consisting of 120 min at 31.5 lb/in2 and 275 degrees F and 75 min at 45 lb/in2 and 292 degrees F effectively decontaminated the BDR material. Two sequential standard autoclave cycles consisting of 40 min at 31.5 lb/in2 and 275 degrees F proved to be particularly effective, probably because the second cycle's evacuation step pulled the condensed water out of the pores of the materials, allowing better steam penetration. The results also indicated that the packing density and material type of the BDR in the autoclave could have a significant impact on the effectiveness of the decontamination process.

[Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

PubMed

Gao, Zhiqi; Wang, Dongshu; Feng, Erling; Wang, Bingxiang; Hui, Yiming; Han, Shaobo; Jiao, Lei; Liu, Xiankai; Wang, Hengliang

2014-11-04

To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. We did not find any polymorphism of CRISPR in B. anthracis. The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

Adherence to Antimicrobial Inhalational Anthrax Prophylaxis among Postal Workers, Washington, D.C., 2001

PubMed Central

Laserson, Kayla; Fry, Alicia M.; Roy, Sharon; Hayslett, James; Grummer-Strawn, Laurence; Kettel-Khan, Laura; Schuchat, Anne

2002-01-01

In October 2001, two envelopes containing Bacillus anthracis spores were processed at the Washington, D.C., Processing and Distribution Center of the U.S. Postal Service; inhalational anthrax developed in four workers at this facility. More than 2,000 workers were advised to complete 60 days of postexposure prophylaxis to prevent inhalational anthrax. Interventions to promote adherence were carried out to support workers, and qualitative information was collected to evaluate our interventions. A quantitative survey was administered to a convenience sample of workers to assess factors influencing adherence. No anthrax infections developed in any workers involved in the interventions or interviews. Of 245 workers, 98 (40%) reported full adherence to prophylaxis, and 45 (18%) had completely discontinued it. Experiencing adverse effects to prophylaxis, anxiety, and being <45 years old were risk factors for discontinuing prophylaxis. Interventions, especially frequent visits by public health staff, proved effective in supporting adherence. PMID:12396929

Lessons to be Learned from Recent Biosafety Incidents in the United States.

PubMed

Weiss, Shay; Yitzhaki, Shmuel; Shapira, Shmuel C

2015-05-01

During recent months, the Centers for Disease Control and Prevention (CDC) announced the occurrence of three major biosafety incidents, raising serious concern about biosafety and biosecurity guideline implementation in the most prestigious agencies in the United States: the CDC, the National Institutes of Health (NIH) and the Federal Drug Administration (FDA). These lapses included: a) the mishandling of Bacillus anthracis spores potentially exposing dozens of employees to anthrax; b) the shipment of low pathogenic influenza virus unknowingly cross-contaminated with a highly pathogenic strain; and c) an inventory lapse of hundreds of samples of biological agents, including six vials of variola virus kept in a cold storage room for decades, unnoticed. In this review we present the published data on these events, report the CDC inquiry's main findings, and discuss the key lessons to be learnt to ensure safer scientific practice in biomedical and microbiological service and research laboratories.

Anthrax Pathogenesis.

PubMed

Moayeri, Mahtab; Leppla, Stephen H; Vrentas, Catherine; Pomerantsev, Andrei P; Liu, Shihui

2015-01-01

Anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis. The bacterium's major virulence factors are (a) the anthrax toxins and (b) an antiphagocytic polyglutamic capsule. These are encoded by two large plasmids, the former by pXO1 and the latter by pXO2. The expression of both is controlled by the bicarbonate-responsive transcriptional regulator, AtxA. The anthrax toxins are three polypeptides-protective antigen (PA), lethal factor (LF), and edema factor (EF)-that come together in binary combinations to form lethal toxin and edema toxin. PA binds to cellular receptors to translocate LF (a protease) and EF (an adenylate cyclase) into cells. The toxins alter cell signaling pathways in the host to interfere with innate immune responses in early stages of infection and to induce vascular collapse at late stages. This review focuses on the role of anthrax toxins in pathogenesis. Other virulence determinants, as well as vaccines and therapeutics, are briefly discussed.

Modeling Tool for Decision Support during Early Days of an Anthrax Event.

PubMed

Rainisch, Gabriel; Meltzer, Martin I; Shadomy, Sean; Bower, William A; Hupert, Nathaniel

2017-01-01

Health officials lack field-implementable tools for forecasting the effects that a large-scale release of Bacillus anthracis spores would have on public health and hospitals. We created a modeling tool (combining inhalational anthrax caseload projections based on initial case reports, effects of variable postexposure prophylaxis campaigns, and healthcare facility surge capacity requirements) to project hospitalizations and casualties from a newly detected inhalation anthrax event, and we examined the consequences of intervention choices. With only 3 days of case counts, the model can predict final attack sizes for simulated Sverdlovsk-like events (1979 USSR) with sufficient accuracy for decision making and confirms the value of early postexposure prophylaxis initiation. According to a baseline scenario, hospital treatment volume peaks 15 days after exposure, deaths peak earlier (day 5), and recovery peaks later (day 23). This tool gives public health, hospital, and emergency planners scenario-specific information for developing quantitative response plans for this threat.

Integration of Stable Isotope and other Mass Spectral Data for Microbial Forensics

NASA Astrophysics Data System (ADS)

Kreuzer-Martin, H. W.; Jarman, K. H.

2008-12-01

The nascent field of microbial forensics requires the development of diverse signatures as indicators of various aspects of the production environment of microorganisms. We have characterized isotopic relationships between Bacillus subtilis ATCC 6051 spores and their growth environment, using as a database the carbon, nitrogen, oxygen and hydrogen stable isotope ratios of a total of 247 separate cultures of spores produced on a total of 32 different culture media. We have analyzed variation within individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times in the context of using stable isotope ratios as a signature for sample matching. We have correlated the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen of growth medium nutrients or water and spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures. The power of stable isotope ratio data can be greatly enhanced by combining it with orthogonal datasets that speak to different aspects of an organism's production environment. We developed a Bayesian network that follows the causal relationship from culture medium recipe to spore elemental content as measured by secondary ion mass spectrometry (SIMS), carbon and nitrogen stable isotope ratios, and to the presence of residual agar by electrospray ionization MS (ESI-MS). The network was developed and tested on data from three replicate cultures of B. subtilis ATCC 49760 in broth and agar-containing versions of four different nutrient media. To test the network, data from SIMS analyses of B. subtilis 49760 produced in a different medium, from approximately 200 ESI MS analyses of B. thuringensis ATCC 58890 and B. anthracis Sterne grown in five additional media, and the stable isotope data from the 247 cultures of B. subtilis 6051 spores were used. This network was able to characterize Bacillus spores grown under multiple culture conditions with an error rate of less than 0.07 in characterizing carbon and nitrogen source, addition of metals, and presence of agar, and an error rate of 0.19 in characterizing the culture medium recipe. The integration of multiple analytical techniques allowed us to maximize the amount of information obtained from unknown source microorganisms. The Bayesian network approach allowed us to combine scientific understanding with well established statistical methodologies to characterize a microbe's growth environment without the need for reference signatures. Similar approaches could be applied to data from other scientific disciplines, as well as to other problems of attribution.

Testing large volume water treatment and crude oil ...

EPA Pesticide Factsheets

Report EPA’s Homeland Security Research Program (HSRP) partnered with the Idaho National Laboratory (INL) to build the Water Security Test Bed (WSTB) at the INL test site outside of Idaho Falls, Idaho. The WSTB was built using an 8-inch (20 cm) diameter cement-mortar lined drinking water pipe that was previously taken out of service. The pipe was exhumed from the INL grounds and oriented in the shape of a small drinking water distribution system. Effluent from the pipe is captured in a lagoon. The WSTB can support drinking water distribution system research on a variety of drinking water treatment topics including biofilms, water quality, sensors, and homeland security related contaminants. Because the WSTB is constructed of real drinking water distribution system pipes, research can be conducted under conditions similar to those in a real drinking water system. In 2014, WSTB pipe was experimentally contaminated with Bacillus globigii spores, a non-pathogenic surrogate for the pathogenic B. anthracis, and then decontaminated using chlorine dioxide. In 2015, the WSTB was used to perform the following experiments: • Four mobile disinfection technologies were tested for their ability to disinfect large volumes of biologically contaminated “dirty” water from the WSTB. B. globigii spores acted as the biological contaminant. The four technologies evaluated included: (1) Hayward Saline C™ 6.0 Chlorination System, (2) Advanced Oxidation Process (A

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar.

PubMed

Tomaso, Herbert; Bartling, Carsten; Al Dahouk, Sascha; Hagen, Ralf M; Scholz, Holger C; Beyer, Wolfgang; Neubauer, Heinrich

2006-01-01

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.

A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax.

PubMed

Gorantala, Jyotsna; Grover, Sonam; Goel, Divya; Rahi, Amit; Jayadev Magani, Sri Krishna; Chandra, Subhash; Bhatnagar, Rakesh

2011-06-15

The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10(4) were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10(5). All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in highlighting the efficacy of plant expressed PA(dIV) by oral immunization in murine model. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protozoal Digestion of Coat-Defective Bacillus subtilis Spores Produces “Rinds” Composed of Insoluble Coat Protein▿

PubMed Central

Carroll, Alicia Monroe; Plomp, Marco; Malkin, Alexander J.; Setlow, Peter

2008-01-01

The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon. PMID:18689521

Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

DTIC Science & Technology

2011-09-01

future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

Resistance to and killing by the sporicidal microbicide peracetic acid.

PubMed

Leggett, Mark J; Schwarz, J Spencer; Burke, Peter A; Mcdonnell, Gerald; Denyer, Stephen P; Maillard, Jean-Yves

2015-03-01

To elucidate the mechanisms of spore resistance to and killing by the oxidizing microbicide peracetic acid (PAA). Mutants of Bacillus subtilis lacking specific spore structures were used to identify resistance properties in spores and to understand the mechanism of action of PAA. We also assessed the effect of PAA treatment on a number of spore properties including heat tolerance, membrane integrity and germination. The spore coat is essential for spore PAA resistance as spores with defective coats were greatly sensitized to PAA treatment. Small acid-soluble spore proteins apparently provide no protection against PAA. Defects in spore germination, specifically in germination via the GerB and GerK but not the GerA germination receptors, as well as leakage of internal components suggest that PAA is active at the spore inner membrane. It is therefore likely that the inner membrane is the major site of PAA's sporicidal activity. PAA treatment targets the spore membrane, with some of its activity directed specifically against the GerB and GerK germination receptors. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge

PubMed Central

Klas, S.D.; Petrie, C.R.; Warwood, S.J.; Williams, M.S.; Olds, C.L.; Stenz, J.P.; Cheff, A.M.; Hinchcliffe, M.; Richardson, C.; Wimer, S.

2009-01-01

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150 μg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150 μg rPA, 150 μg rPA + 150 μg of a conjugated 10-mer peptide representing the B. anthracis capsule (conj), or 150 μg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys®). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA + conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p = 0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA + conj immunized groups (p = 0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later. PMID:18703110

A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge.

PubMed

Klas, S D; Petrie, C R; Warwood, S J; Williams, M S; Olds, C L; Stenz, J P; Cheff, A M; Hinchcliffe, M; Richardson, C; Wimer, S

2008-10-09

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150microg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150microg rPA, 150microg rPA+150microg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150microg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.

Rapid filtration separation-based sample preparation method for Bacillus spores in powdery and environmental matrices.

PubMed

Isabel, Sandra; Boissinot, Maurice; Charlebois, Isabelle; Fauvel, Chantal M; Shi, Lu-E; Lévesque, Julie-Christine; Paquin, Amélie T; Bastien, Martine; Stewart, Gale; Leblanc, Eric; Sato, Sachiko; Bergeron, Michel G

2012-03-01

Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.

Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

PubMed Central

Isabel, Sandra; Boissinot, Maurice; Charlebois, Isabelle; Fauvel, Chantal M.; Shi, Lu-E; Lévesque, Julie-Christine; Paquin, Amélie T.; Bastien, Martine; Stewart, Gale; Leblanc, Éric; Sato, Sachiko

2012-01-01

Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation. PMID:22210204

Screening of Peptide Libraries Against Protective Antigen of Bacillus Anthracis in a Disposable Microfluidic Cartridge

DTIC Science & Technology

2011-11-28

New Reprint Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge W911NF-09-D-0001...against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Report Title ABSTRACT See attached. Screening of Peptide...Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Joshua M. Kogot1, Yanting Zhang2, Stephen J. Moore3

Draft Genome Sequences from a Novel Clade of Bacillus cereus Sensu Lato Strains, Isolated from the International Space Station.

PubMed

Venkateswaran, Kasthuri; Checinska Sielaff, Aleksandra; Ratnayake, Shashikala; Pope, Robert K; Blank, Thomas E; Stepanov, Victor G; Fox, George E; van Tongeren, Sandra P; Torres, Clinton; Allen, Jonathan; Jaing, Crystal; Pierson, Duane; Perry, Jay; Koren, Sergey; Phillippy, Adam; Klubnik, Joy; Treangen, Todd J; Rosovitz, M J; Bergman, Nicholas H

2017-08-10

The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis - B. cereus - B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three strains), and Russian Segment Zvezda Module (two strains). Copyright © 2017 Venkateswaran et al.

Evaluating Composite Sampling Methods of Bacillus Spores at Low Concentrations

PubMed Central

Hess, Becky M.; Amidan, Brett G.; Anderson, Kevin K.; Hutchison, Janine R.

2016-01-01

Restoring all facility operations after the 2001 Amerithrax attacks took years to complete, highlighting the need to reduce remediation time. Some of the most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite (SM-SPC): a single cellulose sponge samples multiple coupons with a single pass across each coupon; 2) single medium multi-pass composite: a single cellulose sponge samples multiple coupons with multiple passes across each coupon (SM-MPC); and 3) multi-medium post-sample composite (MM-MPC): a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155 CFU/cm2). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted dry wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p< 0.0001) and coupon material (p = 0.0006). Recovery efficiency (RE) was higher overall using the MM-MPC method compared to the SM-SPC and SM-MPC methods. RE with the MM-MPC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, dry wall, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when responding to a Bacillus anthracis contamination event of clean or dirty surfaces. PMID:27736999

Evaluating Composite Sampling Methods of Bacillus Spores at Low Concentrations.

PubMed

Hess, Becky M; Amidan, Brett G; Anderson, Kevin K; Hutchison, Janine R

2016-01-01

Restoring all facility operations after the 2001 Amerithrax attacks took years to complete, highlighting the need to reduce remediation time. Some of the most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite (SM-SPC): a single cellulose sponge samples multiple coupons with a single pass across each coupon; 2) single medium multi-pass composite: a single cellulose sponge samples multiple coupons with multiple passes across each coupon (SM-MPC); and 3) multi-medium post-sample composite (MM-MPC): a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155 CFU/cm2). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted dry wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p< 0.0001) and coupon material (p = 0.0006). Recovery efficiency (RE) was higher overall using the MM-MPC method compared to the SM-SPC and SM-MPC methods. RE with the MM-MPC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, dry wall, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when responding to a Bacillus anthracis contamination event of clean or dirty surfaces.

Evaluating Composite Sampling Methods of Bacillus spores at Low Concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hess, Becky M.; Amidan, Brett G.; Anderson, Kevin K.

Restoring facility operations after the 2001 Amerithrax attacks took over three months to complete, highlighting the need to reduce remediation time. The most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite: a single cellulose sponge samples multiple coupons; 2) single medium multi-pass composite: a single cellulose sponge is used to sample multiple coupons; and 3) multi-medium post-samplemore » composite: a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155CFU/cm2, respectively). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p-value < 0.0001) and coupon material (p-value = 0.0008). Recovery efficiency (RE) was higher overall using the post-sample composite (PSC) method compared to single medium composite from both clean and grime coated materials. RE with the PSC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, painted wall board, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but significantly lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when responding to a Bacillus anthracis contamination event of clean or dirty surfaces.« less

Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

PubMed

Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

2016-01-06

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The dark sides of capillary morphogenesis gene 2

PubMed Central

Deuquet, Julie; Lausch, Ekkehart; Superti-Furga, Andrea; van der Goot, F Gisou

2012-01-01

Capillary morphogenesis gene 2 (CMG2) is a type I membrane protein involved in the homeostasis of the extracellular matrix. While it shares interesting similarities with integrins, its exact molecular role is unknown. The interest and knowledge about CMG2 largely stems from the fact that it is involved in two diseases, one infectious and one genetic. CMG2 is the main receptor of the anthrax toxin, and knocking out this gene in mice renders them insensitive to infection with Bacillus anthracis spores. On the other hand, mutations in CMG2 lead to a rare but severe autosomal recessive disorder in humans called Hyaline Fibromatosis Syndrome (HFS). We will here review what is known about the structure of CMG2 and its ability to mediate anthrax toxin entry into cell. We will then describe the limited knowledge available concerning the physiological role of CMG2. Finally, we will describe HFS and the consequences of HFS-associated mutations in CMG2 at the molecular and cellular level. PMID:22215446

A systematic methodology for selecting decontamination strategies following a biocontamination event.

PubMed

Krauter, Paula; Edwards, Donna; Yang, Lynn; Tucker, Mark

2011-09-01

Decontamination and recovery of a facility or outdoor area after a wide-area biological incident involving a highly persistent agent (eg, Bacillus anthracis spores) is a complex process that requires extensive information and significant resources, which are likely to be limited, particularly if multiple facilities or areas are affected. This article proposes a systematic methodology for evaluating information to select the decontamination or alternative treatments that optimize use of resources if decontamination is required for the facility or area. The methodology covers a wide range of approaches, including volumetric and surface decontamination, monitored natural attenuation, and seal and abandon strategies. A proposed trade-off analysis can help decision makers understand the relative appropriateness, efficacy, and labor, skill, and cost requirements of the various decontamination methods for the particular facility or area needing treatment--whether alone or as part of a larger decontamination effort. Because the state of decontamination knowledge and technology continues to evolve rapidly, the methodology presented here is designed to accommodate new strategies and materials and changing information.

Clostridial binary toxins: iota and C2 family portraits.

PubMed

Stiles, Bradley G; Wigelsworth, Darran J; Popoff, Michel R; Barth, Holger

2011-01-01

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host-cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium.

Anthrax: Where Margins are Merging between Emerging Threats and Bioterrorism

PubMed Central

Banerjee, Dibyendu; Chakraborty, Baishali; Chakraborty, Banya

2017-01-01

National Institute of Allergy and Infectious Diseases has classified all the emerging infectious diseases agents under three categories. Among Category A priority pathogens comes Bacillus anthracis –the causative agent of Anthrax. It is a gram positive spore bearing bacteria, and the disease is typically associated with grazing animals, and affects the people as a zoonosis. The disease can be classically transmitted by three routes namely: cutaneous, gastrointestinal and pulmonary, with a fourth route recently identified as “injection anthrax”, seen in intravenous drug abusers. Cutaneous anthrax is the commonest form in humans, accounting for 95% of all the cases. There are two main virulence factors of this bacteria, a capsule and an exotoxin, each carried by a separate toxin. Two models have been used for explaining the pathogenesis of this infection. The earlier one or “Trojan horse” model is now replaced with “jail-break” model. Centers for disease control (CDC) has issued updated guidelines for diagnosis, post-exposure prophylaxis and treatment. For immunization, anthrax vaccine absorbed is available. PMID:28979006

Correlation between anthrax lethal toxin neutralizing antibody levels and survival in guinea pigs and nonhuman primates vaccinated with the AV7909 anthrax vaccine candidate.

PubMed

Savransky, Vladimir; Shearer, Jeffry D; Gainey, Melicia R; Sanford, Daniel C; Sivko, Gloria S; Stark, Gregory V; Li, Na; Ionin, Boris; Lacy, Michael J; Skiadopoulos, Mario H

2017-09-05

The anthrax vaccine candidate AV7909 is being developed as a next generation vaccine for a post-exposure prophylaxis (PEP) indication against anthrax. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA, BioThrax®) bulk drug substance adjuvanted with the immunostimulatory oligodeoxynucleotide (ODN) compound, CPG 7909. The addition of CPG 7909 to AVA enhances both the magnitude and the kinetics of antibody responses in animals and human subjects, making AV7909 a suitable next-generation vaccine for use in a PEP setting. The studies described here provide initial information on AV7909-induced toxin-neutralizing antibody (TNA) levels associated with the protection of animals from lethal Bacillus anthracis challenge. Guinea pigs or nonhuman primates (NHPs) were immunized on Days 0 and 28 with various dilutions of AV7909, AVA or a saline or Alhydrogel+CPG 7909 control. Animals were challenged via the inhalational route with a lethal dose of aerosolized B. anthracis (Ames strain) spores and observed for clinical signs of disease and mortality. The relationship between pre-challenge serum TNA levels and survival following challenge was determined in order to calculate a threshold TNA level associated with protection. Immunisation with AV7909 induced a rapid, highly protective TNA response in guinea pigs and NHPs. Surprisingly, the TNA threshold associated with a 70% probability of survival for AV7909 immunized animals was substantially lower than the threshold which has been established for the licensed AVA vaccine. The results of this study suggest that the TNA threshold of protection against anthrax could be modified by the addition of an immune stimulant such as CPG 7909 and that the TNA levels associated with protection may be vaccine-specific. Copyright © 2017. Published by Elsevier Ltd.

Evaluation of a plasmid DNA-based anthrax vaccine in rabbits, nonhuman primates and healthy adults.

PubMed

Keitel, Wendy A; Treanor, John J; El Sahly, Hana M; Evans, Thomas G; Kopper, Scott; Whitlow, Vanessa; Selinsky, Cheryl; Kaslow, David C; Rolland, Alain; Smith, Larry R; Lalor, Peggy A

2009-08-01

VCL-AB01, a cationic lipid-formulated plasmid DNA (pDNA)-based vaccine that contains genes encoding genetically detoxified Bacillus anthracis protective antigen (PA) and lethal factor (LF), was assessed in a Phase 1, dose-escalating clinical trial in healthy adults for safety and immunogenicity, and in nonhuman primates for immunogenicity and efficacy against challenge with a lethal dose of B. anthracis spores. Healthy 18-45 year old subjects were randomly assigned to receive either the investigational vaccine containing 0.2 mg, 0.6 mg, or 2 mg of total pDNA per dose, or saline placebo, administered at 0, 1 and 2 months. The 0.2 mg and 0.6 mg dose levels were generally well tolerated; however, dose-limiting reactogenicity was observed among subjects given the first 2 mg dose and the remaining two injections in the 2 mg group were reduced to 0.6 mg. Dose-related increases in seroconversion frequencies were observed. Overall, 10%, 33.3% and 80% of subjects in the 0.2, 0.6 and 2 mg groups, respectively, developed antibodies to PA and/or LF as measured by ELISA; however, antibodies with toxin neutralizing activity (TNA) were detected in only one subject. In monkeys that received a 0.6 mg dose three times at 2 week intervals, low levels of antibodies were detected by ELISA but not by the TNA assay in all animals just prior to challenge. Despite the absence of TNA, 75% animals survived the lethal challenge. In summary, VCL-AB01 was generally well tolerated in humans at a dose that provided immunity in monkeys despite the lack of robust TNA titers in either species.

Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence ◊

PubMed Central

Stoddard, Robyn A.; Quinn, Conrad P.; Schiffer, Jarad M.; Boyer, Anne E.; Goldstein, Jason; Bagarozzi, Dennis A.; Soroka, Stephen D.; Dauphin, Leslie A.; Hoffmaster, Alex R.

2015-01-01

Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10−6 μM (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

Development of an Inhalational Bacillus anthracis Exposure Therapeutic Model in Cynomolgus Macaques

PubMed Central

Comer, Jason E.; Stark, Gregory V.; Ray, Bryan D.; Tordoff, Kevin P.; Knostman, Katherine A. B.; Meister, Gabriel T.

2012-01-01

Appropriate animal models are required to test medical countermeasures to bioterrorist threats. To that end, we characterized a nonhuman primate (NHP) inhalational anthrax therapeutic model for use in testing anthrax therapeutic medical countermeasures according to the U.S. Food and Drug Administration Animal Rule. A clinical profile was recorded for each NHP exposed to a lethal dose of Bacillus anthracis Ames spores. Specific diagnostic parameters were detected relatively early in disease progression, i.e., by blood culture (∼37 h postchallenge) and the presence of circulating protective antigen (PA) detected by electrochemiluminescence (ECL) ∼38 h postchallenge, whereas nonspecific clinical signs of disease, i.e., changes in body temperature, hematologic parameters (ca. 52 to 66 h), and clinical observations, were delayed. To determine whether the presentation of antigenemia (PA in the blood) was an appropriate trigger for therapeutic intervention, a monoclonal antibody specific for PA was administered to 12 additional animals after the circulating levels of PA were detected by ECL. Seventy-five percent of the monoclonal antibody-treated animals survived compared to 17% of the untreated controls, suggesting that intervention at the onset of antigenemia is an appropriate treatment trigger for this model. Moreover, the onset of antigenemia correlated with bacteremia, and NHPs were treated in a therapeutic manner. Interestingly, brain lesions were observed by histopathology in the treated nonsurviving animals, whereas this observation was absent from 90% of the nonsurviving untreated animals. Our results support the use of the cynomolgus macaque as an appropriate therapeutic animal model for assessing the efficacy of medical countermeasures developed against anthrax when administered after a confirmation of infection. PMID:22956657

Anthrax Vaccine Precipitated Induces Edema Toxin-Neutralizing, Edema Factor-Specific Antibodies in Human Recipients

PubMed Central

Dumas, Eric K.; Gross, Timothy; Larabee, Jason; Pate, Lance; Cuthbertson, Hannah; Charlton, Sue; Hallis, Bassam; Engler, Renata J. M.; Collins, Limone C.; Spooner, Christina E.; Chen, Hua; Ballard, Jimmy; James, Judith A.

2017-01-01

ABSTRACT Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP (n = 33) versus anthrax vaccine adsorbed (AVA; n = 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 ± 58.6) of EF antibodies than AVA (4.2% and 7.8 ± 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines. PMID:28877928

Cross-species prediction of human survival probabilities for accelerated anthrax vaccine absorbed (AVA) regimens and the potential for vaccine and antibiotic dose sparing.

PubMed

Stark, G V; Sivko, G S; VanRaden, M; Schiffer, J; Taylor, K L; Hewitt, J A; Quinn, C P; Nuzum, E O

2016-12-12

Anthrax vaccine adsorbed (AVA, BioThrax) was recently approved by the Food and Drug Administration (FDA) for a post-exposure prophylaxis (PEP) indication in adults 18-65years of age. The schedule is three doses administered subcutaneous (SC) at 2-week intervals (0, 2, and 4weeks), in conjunction with a 60-day course of antimicrobials. The Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) developed an animal model to support assessment of a shortened antimicrobial PEP duration following Bacillus anthracis exposure. A nonhuman primate (NHP) study was completed to evaluate the efficacy of a two dose anthrax vaccine absorbed (AVA) schedule (0, 2weeks) aerosol challenged with high levels of B. anthracis spores at week4- the time point at which humans would receive the third vaccination of the approved PEP schedule. Here we use logistic regression models to combine the survival data from the NHP study along with serum anthrax lethal toxin neutralizing activity (TNA) and anti-PA IgG measured by enzyme linked immunosorbent assay (ELISA) data to perform a cross-species analysis to estimate survival probabilities in vaccinated human populations at this time interval (week4 of the PEP schedule). The bridging analysis demonstrated that high levels of NHP protection also yield high predicted probability of human survival just 2weeks after the second dose of vaccine with the full or half antigen dose regimen. The absolute difference in probability of human survival between the full and half antigen dose was estimated to be at most approximately 20%, indicating that more investigation of the half-antigen dose for vaccine dose sparing strategies may be warranted. Copyright © 2016 Elsevier Ltd. All rights reserved.

Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

PubMed

Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

2016-08-01

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genetic Characterization of Bacillus anthracis 17 JB strain.

PubMed

Seyed-Mohamadi, Sakineh; Moradi Bidhendi, Soheila; Tadayon, Keyvan; Ghaderi, Rainak

2015-06-01

Bacillus anthracis is one of the most homogenous bacteria ever described. Some level of diversity. Bacillus anthracis 17JB is a laboratory strain It is broadly used as a challenge strain in guinea pigs for potency test of anthrax vaccine. This work describes genetic characterization of B. anthracis 17 JB strain using the SNPs and MLVA genotyping. In SNPs typing, the originally French 17JB strain represented the A.Br. 008/009 subgroup. In Levy's genotyping method, 843, 451 and 864 bp long fragments were identified at AA03, AJ03 and AA07 loci, respectively. In the vaccine manufacturer perspective these findings are much valuable on their own account, but similar research is required to extend molecular knowledge of B. anthracis epidemiology in Persia.

Detection of biological warfare agents using ultra violet-laser induced fluorescence LIDAR

NASA Astrophysics Data System (ADS)

Joshi, Deepti; Kumar, Deepak; Maini, Anil K.; Sharma, Ramesh C.

This review has been written to highlight the threat of biological warfare agents, their types and detection. Bacterial biological agent Bacillus anthracis (bacteria causing the disease anthrax) which is most likely to be employed in biological warfare is being discussed in detail. Standoff detection of biological warfare agents in aerosol form using Ultra violet-Laser Induced Fluorescence (UV-LIF) spectroscopy method has been studied. Range-resolved detection and identification of biological aerosols by both nano-second and non-linear femto-second LIDAR is also discussed. Calculated received fluorescence signal for a cloud of typical biological agent Bacillus globigii (Simulants of B. anthracis) at a location of ˜5.0 km at different concentrations in presence of solar background radiation has been described. Overview of current research efforts in internationally available working UV-LIF LIDAR systems are also mentioned briefly.

The Pathogenomic Sequence Analysis of B. cereus and B. Thuringiensis isolates closely related to Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, C S; Xie, G; Challacombe, J F

The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomesmore » with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.« less

Specific identification of Bacillus anthracis strains

NASA Astrophysics Data System (ADS)

Krishnamurthy, Thaiya; Deshpande, Samir; Hewel, Johannes; Liu, Hongbin; Wick, Charles H.; Yates, John R., III

2007-01-01

Accurate identification of human pathogens is the initial vital step in treating the civilian terrorism victims and military personnel afflicted in biological threat situations. We have applied a powerful multi-dimensional protein identification technology (MudPIT) along with newly generated software termed Profiler to identify the sequences of specific proteins observed for few strains of Bacillus anthracis, a human pathogen. Software termed Profiler was created to initially screen the MudPIT data of B. anthracis strains and establish the observed proteins specific for its strains. A database was also generated using Profiler containing marker proteins of B. anthracis and its strains, which in turn could be used for detecting the organism and its corresponding strains in samples. Analysis of the unknowns by our methodology, combining MudPIT and Profiler, led to the accurate identification of the anthracis strains present in samples. Thus, a new approach for the identification of B. anthracis strains in unknown samples, based on the molecular mass and sequences of marker proteins, has been ascertained.

Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

PubMed

Sharma, Mukesh Kumar; Narayanan, J; Pardasani, Deepak; Srivastava, Divesh N; Upadhyay, Sanjay; Goel, Ajay Kumar

2016-06-15

Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrazine vapor inactivates Bacillus spores

NASA Astrophysics Data System (ADS)

Schubert, Wayne W.; Engler, Diane L.; Beaudet, Robert A.

2016-05-01

NASA policy restricts the total number of bacterial spores that can remain on a spacecraft traveling to any planetary body which might harbor life or have evidence of past life. Hydrazine, N2H4, is commonly used as a propellant on spacecraft. Hydrazine as a liquid is known to inactivate bacterial spores. We have now verified that hydrazine vapor also inactivates bacterial spores. After Bacillus atrophaeus ATCC 9372 spores deposited on stainless steel coupons were exposed to saturated hydrazine vapor in closed containers, the spores were recovered from the coupons, serially diluted, pour plated and the surviving bacterial colonies were counted. The exposure times required to reduce the spore population by a factor of ten, known as the D-value, were 4.70 ± 0.50 h at 25 °C and 2.85 ± 0.13 h at 35 °C. These inactivation rates are short enough to ensure that the bioburden of the surfaces and volumes would be negligible after prolonged exposure to hydrazine vapor. Thus, all the propellant tubing and internal tank surfaces exposed to hydrazine vapor do not contribute to the total spore count.

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

PubMed Central

Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

2007-01-01

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate. PMID:17720820

Ten Genome Sequences of Human and Livestock Isolates of Bacillus anthracis from the Country of Georgia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khmaladze, Ekaterine; Dzavashvili, Giorgi; Chanturia, Gvantsa

Bacillus anthracis causes the acute fatal disease anthrax, is a proven biological weapon, and is endemic in Georgia, where human and animal cases are reported annually. Furthermore, we present whole-genome sequences of 10 historical B. anthracis strains from Georgia.

Ten Genome Sequences of Human and Livestock Isolates of Bacillus anthracis from the Country of Georgia

DOE PAGES

Khmaladze, Ekaterine; Dzavashvili, Giorgi; Chanturia, Gvantsa; ...

2017-05-11

Bacillus anthracis causes the acute fatal disease anthrax, is a proven biological weapon, and is endemic in Georgia, where human and animal cases are reported annually. Furthermore, we present whole-genome sequences of 10 historical B. anthracis strains from Georgia.

Assessment of a Solid Phase Matrix for the Neutralization and Real-Time PCR Detection of Bacillus anthracis

DTIC Science & Technology

2006-12-01

marcb6, a & 6valu~e pour sa capacitd A neutraliser le Bacillus anthracis vivant et A trapper l’acide nuclkide et effectuer une analyse g~n~tique au moyen... vivant indique que les plaques ETA® neutralisaient les B. anthracis vivants mais A des concentrations faibles. C’est pourquoi on doit estimer que les...plaques FTA® macul~es d𔄀chantillons contenant ou suspects de contenir du B. anthracis vivant sont potentiellement infectieuses. Les analyses PCR des

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

PubMed Central

Nattermann, Herbert; Brüggemann, Holger; Dupke, Susann; Wollherr, Antje; Franz, Tatjana; Pauli, Georg; Appel, Bernd; Liebl, Wolfgang; Couacy-Hymann, Emmanuel; Boesch, Christophe; Meyer, Frauke-Dorothee; Leendertz, Fabian H.; Ellerbrok, Heinz; Gottschalk, Gerhard; Grunow, Roland; Liesegang, Heiko

2010-01-01

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”. PMID:20634886

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

PubMed Central

Be, Nicholas A.; Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Fofanov, Viacheslav Y.; Koshinsky, Heather; Ellingson, Sally R.; Brettin, Thomas S.; Jackson, Paul J.; Jaing, Crystal J.

2013-01-01

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. PMID:24039948

Bacillus anthracis virulence in Guinea pigs vaccinated with anthrax vaccine adsorbed is linked to plasmid quantities and clonality.

PubMed

Coker, Pamala R; Smith, Kimothy L; Fellows, Patricia F; Rybachuck, Galena; Kousoulas, Konstantin G; Hugh-Jones, Martin E

2003-03-01

Bacillus anthracis is a bacterial pathogen of great importance, both historically and in the present. This study presents data collected from several investigations and indicates that B. anthracis virulence is associated with the clonality and virulence of plasmids pXO1 and pXO2. Guinea pigs vaccinated with Anthrax Vaccine Adsorbed were challenged with 20 B. anthracis isolates representative of worldwide genetic diversity. These same isolates were characterized with respect to plasmid copy number by using a novel method of quantitative PCR developed for rapid and efficient detection of B. anthracis from environmental samples. We found that the copy numbers for both pXO1 and pXO2 differed from those in previously published reports. By combining the data on survival, plasmid copy numbers, and clonality, we developed a model predicting virulence. This model was validated by using a randomly chosen set of 12 additional B. anthracis isolates. Results from this study will be helpful in future efforts to elucidate the basis for variation in the virulence of this important pathogen.

Application of Pyrosequencing® in Food Biodefense.

PubMed

Amoako, Kingsley Kwaku

2015-01-01

The perpetration of a bioterrorism attack poses a significant risk for public health with potential socioeconomic consequences. It is imperative that we possess reliable assays for the rapid and accurate identification of biothreat agents to make rapid risk-informed decisions on emergency response. The development of advanced methodologies for the detection of biothreat agents has been evolving rapidly since the release of the anthrax spores in the mail in 2001, and recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence-based approaches such as Pyrosequencing(®), which has the capability to determine short DNA stretches in real time using biotinylated PCR amplicons, have potential biodefense applications. Using markers from the virulence plasmids and chromosomal regions, my laboratory has demonstrated the power of this technology in the rapid, specific, and sensitive detection of B. anthracis spores and Yersinia pestis in food. These are the first applications for the detection of the two organisms in food. Furthermore, my lab has developed a rapid assay to characterize the antimicrobial resistance (AMR) gene profiles for Y. pestis using Pyrosequencing. Pyrosequencing is completed in about 60 min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence, thus enabling rapid risk-informed decisions to be made. A typical run yields 40-84 bp reads with 94-100 % identity to the expected sequence. It also provides a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The method described is proposed as a novel detection system for potential application in food biodefense.

Mapping as a tool for predicting the risk of anthrax outbreaks in Northern Region of Ghana.

PubMed

Nsoh, Ayamdooh Evans; Kenu, Ernest; Forson, Eric Kofi; Afari, Edwin; Sackey, Samuel; Nyarko, Kofi Mensah; Yebuah, Nathaniel

2016-01-01

Anthrax is a febrile soil-born infectious disease that can affect all warm-blooded animals including man. Outbreaks of anthrax have been reported in northern region of Ghana but no concerted effort has been made to implement risk-based surveillance systems to document outbreaks so as to implement policies to address the disease. We generated predictive maps using soil pH, temperature and rainfall as predictor variables to identify hotspot areas for the outbreaks. A 10-year secondary data records on soil pH, temperature and rainfall were used to create climate-based risk maps using ArcGIS 10.2. The monthly mean values of rainfall and temperature for ten years were calculated and anthrax related evidence based constant raster values were created as weights for the three factors. All maps were generated using the Kriging interpolation method. There were 43 confirmed outbreaks. The deaths involved were 131 cattle, 44 sheep, 15 goats, 562 pigs with 6 human deaths and 22 developed cutaneous anthrax. We found three strata of well delineated distribution pattern indicating levels of risk due to suitability of area for anthrax spore survival. The likelihood of outbreaks occurrence and reoccurrence was higher in Strata I, Strata II and strata III respectively in descending order, due to the suitability of soil pH, temperature and rainfall for the survival and dispersal of B. anthracis spore. The eastern corridor of Northern region is a Hots spot area. Policy makers can develop risk based surveillance system and focus on this area to mitigate anthrax outbreaks and reoccurrence.

Detection of biological warfare agents using ultra violet-laser induced fluorescence LIDAR.

PubMed

Joshi, Deepti; Kumar, Deepak; Maini, Anil K; Sharma, Ramesh C

2013-08-01

This review has been written to highlight the threat of biological warfare agents, their types and detection. Bacterial biological agent Bacillus anthracis (bacteria causing the disease anthrax) which is most likely to be employed in biological warfare is being discussed in detail. Standoff detection of biological warfare agents in aerosol form using Ultra violet-Laser Induced Fluorescence (UV-LIF) spectroscopy method has been studied. Range-resolved detection and identification of biological aerosols by both nano-second and non-linear femto-second LIDAR is also discussed. Calculated received fluorescence signal for a cloud of typical biological agent Bacillus globigii (Simulants of B. anthracis) at a location of ~5.0 km at different concentrations in presence of solar background radiation has been described. Overview of current research efforts in internationally available working UV-LIF LIDAR systems are also mentioned briefly. Copyright © 2013 Elsevier B.V. All rights reserved.

Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beierlein, J.; Frey, K; Bolstad, D

2008-01-01

Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structuremore » of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.« less

Permeability of bacterial spores. IV. Water content, uptake, and distribution.

PubMed

BLACK, S H; GERHARDT, P

1962-05-01

Black, S. H. (The University of Michigan, Ann Arbor) and Philipp Gerhardt. Permeability of bacterial spores. IV. Water content, uptake, and distribution. J. Bacteriol. 83:960-967. 1962.-Dormant and germinated spores of Bacillus cereus strain terminalis were examined for water properties. Respectively, they exhibited a mean density of 1.28 and 1.11 g/ml, a water content of 64.8 and 73.0%, and a total water uptake of 66.6 and 75.6%, based on spore weight, or 86.0 and 83.9%, based on spore volume. The results confirmed a previous report that internal and external water are in virtually complete equilibrium, but refuted a prevailing hypothesis that heat resistance is attributable to a dry core. A model of spore ultrastructure that evolved from the cumulative results pictures a moist, dense, heteroporous core. A new hypothesis is formulated as an explanation for thermostability in spores and possibly in other instances; it postulates the occurrence of an insolubly gelled core with cross-linking between macromolecules through stable but reversible bonds so as to form a high-polymer matrix with entrapped free water.

Decrease in optical density as a results of germination of Alicyclobacillus acidoterrestris spores under high hydrostatic pressure

NASA Astrophysics Data System (ADS)

Porębska, I.; Rutkowska, M.; Sokołowska, B.

2015-01-01

Alicyclobacillus acidoterrestris is a spore-forming bacterium, causing spoilage of juices. The spores of these bacteria have the ability to survive in the typical conditions used for thermal pasteurization. Therefore, the use of other techniques such as high hydrostatic pressure is considered for their inactivation. The effect of hydrostatic pressure of 200-500 MPa, at temperatures 4-50 °C for 15 min, on the dynamics of germination of A. acidoterrestris spores in apple juice and pH 4 buffer was studied. To estimate the share of germinated spores, the method of determining the optical density at a wavelength of 660 nm (OD660) was used. Parameters of hydrostatic pressure treatment used in this work affected the dynamics of germination of A. acidoterrestris spores in apple juice, and the temperature had the greatest effect. The results indicate that nutrients present in apple juice can promote the germination of A. acidoterrestris spores. This paper was presented at the 8th International Conference on High Pressure Bioscience & Biotechnology (HPBB 2014) in Nantes (France) 15-18 July 2014.

Fungal Spores Viability on the International Space Station

NASA Astrophysics Data System (ADS)

Gomoiu, I.; Chatzitheodoridis, E.; Vadrucci, S.; Walther, I.; Cojoc, R.

2016-11-01

In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

Fungal Spores Viability on the International Space Station.

PubMed

Gomoiu, I; Chatzitheodoridis, E; Vadrucci, S; Walther, I; Cojoc, R

2016-11-01

In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.

ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 formore » both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection« less

Background frequency of Bacillus species at the Canberra Airport: A 12 month study.

PubMed

Gahan, Michelle E; Thomas, Rory; Rossi, Rebecca; Nelson, Michelle; Roffey, Paul; Richardson, Michelle M; McNevin, Dennis

2015-12-01

Anthrax, caused by Bacillus anthracis, is a naturally occurring disease in Australia. Whilst mainly limited to livestock in grazing regions of Victoria and New South Wales, movement of people, stock and vehicles means B. anthracis could be present outside this region. Of particular interest is the "background" prevalence of B. anthracis at transport hubs including airports. The aim of this study was to determine the background frequency of B. anthracis and the commonly used hoax agent Bacillus thuringiensis at the Canberra Airport over a 12 month period. Samples were collected daily for seven days each month from August 2011-July 2012 and analyzed using species specific real-time polymerase chain reaction. Fourteen samples (of a total of 575) were positive for the B. anthracis PL3 genomic marker, 24 for the cya (pXO1) plasmid marker and five for the capB (pXO2) plasmid marker. Whilst five samples were positive for both PL3 and cya, no samples were positive for all three markers hence there is no evidence to suggest the presence of pathogenic B. anthracis strains. B. anthracis targets were detected primarily in February 2012 and B. thuringiensis peaked in October and November 2011 and again in April and May 2012. This study provides a rapid method to screen for, and differentiate, Bacillus species. Armed with this information investigators will be able to discriminate a "threat" from "background" frequencies should the need arise. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684.

PubMed

Forsberg, L Scott; Abshire, Teresa G; Friedlander, Arthur; Quinn, Conrad P; Kannenberg, Elmar L; Carlson, Russell W

2012-08-01

Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH(2)-(1→.

Clostridial Binary Toxins: Iota and C2 Family Portraits

PubMed Central

Stiles, Bradley G.; Wigelsworth, Darran J.; Popoff, Michel R.; Barth, Holger

2011-01-01

There are many pathogenic Clostridium species with diverse virulence factors that include protein toxins. Some of these bacteria, such as C. botulinum, C. difficile, C. perfringens, and C. spiroforme, cause enteric problems in animals as well as humans. These often fatal diseases can partly be attributed to binary protein toxins that follow a classic AB paradigm. Within a targeted cell, all clostridial binary toxins destroy filamentous actin via mono-ADP-ribosylation of globular actin by the A component. However, much less is known about B component binding to cell-surface receptors. These toxins share sequence homology amongst themselves and with those produced by another Gram-positive, spore-forming bacterium also commonly associated with soil and disease: Bacillus anthracis. This review focuses upon the iota and C2 families of clostridial binary toxins and includes: (1) basics of the bacterial source; (2) toxin biochemistry; (3) sophisticated cellular uptake machinery; and (4) host–cell responses following toxin-mediated disruption of the cytoskeleton. In summary, these protein toxins aid diverse enteric species within the genus Clostridium. PMID:22919577

Survival of Spores of Trichoderma longibrachiatum in Space: data from the Space Experiment SPORES on EXPOSE-R

NASA Astrophysics Data System (ADS)

Neuberger, Katja; Lux-Endrich, Astrid; Panitz, Corinna

2015-01-01

In the space experiment `Spores in artificial meteorites' (SPORES), spores of the fungus Trichoderma longibrachiatum were exposed to low-Earth orbit for nearly 2 years on board the EXPOSE-R facility outside of the International Space Station. The environmental conditions tested in space were: space vacuum at 10-7-10-4 Pa or argon atmosphere at 105 Pa as inert gas atmosphere, solar extraterrestrial ultraviolet (UV) radiation at λ > 110 nm or λ > 200 nm with fluences up to 5.8 × 108 J m-2, cosmic radiation of a total dose range from 225 to 320 mGy, and temperature fluctuations from -25 to +50°C, applied isolated or in combination. Comparable control experiments were performed on ground. After retrieval, viability of spores was analysed by two methods: (i) ethidium bromide staining and (ii) test of germination capability. About 30% of the spores in vacuum survived the space travel, if shielded against insolation. However, in most cases no significant decrease was observed for spores exposed in addition to the full spectrum of solar UV irradiation. As the spores were exposed in clusters, the outer layers of spores may have shielded the inner part. The results give some information about the likelihood of lithopanspermia, the natural transfer of micro-organisms between planets. In addition to the parameters of outer space, sojourn time in space seems to be one of the limiting parameters.

Transcriptomic responses of germinating Bacillus subtilis spores exposed to 1.5 years of space and simulated martian conditions on the EXPOSE-E experiment PROTECT.

PubMed

Nicholson, Wayne L; Moeller, Ralf; Horneck, Gerda

2012-05-01

Because of their ubiquity and resistance to spacecraft decontamination, bacterial spores are considered likely potential forward contaminants on robotic missions to Mars. Thus, it is important to understand their global responses to long-term exposure to space or martian environments. As part of the PROTECT experiment, spores of B. subtilis 168 were exposed to real space conditions and to simulated martian conditions for 559 days in low-Earth orbit mounted on the EXPOSE-E exposure platform outside the European Columbus module on the International Space Station. Upon return, spores were germinated, total RNA extracted, fluorescently labeled, and used to probe a custom Bacillus subtilis microarray to identify genes preferentially activated or repressed relative to ground control spores. Increased transcript levels were detected for a number of stress-related regulons responding to DNA damage (SOS response, SPβ prophage induction), protein damage (CtsR/Clp system), oxidative stress (PerR regulon), and cell envelope stress (SigV regulon). Spores exposed to space demonstrated a much broader and more severe stress response than spores exposed to simulated martian conditions. The results are discussed in the context of planetary protection for a hypothetical journey of potential forward contaminant spores from Earth to Mars and their subsequent residence on Mars.

The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Cliff S.; Xie, Gary; Challacombe, Jean F.

The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with othermore » members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.« less

On-column labeling of gram-positive bacteria with a boronic acid functionalized squarylium cyanine dye for analysis by polymer-enhanced capillary transient isotachophoresis.

PubMed

Saito, Shingo; Massie, Tara L; Maeda, Takeshi; Nakazumi, Hiroyuki; Colyer, Christa L

2012-03-06

A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ(ex) = 630 nm, λ(em) = 660 nm) with a high affinity constant (K = ~10(2.80) M(-1)) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, Saccharomyces cerevisiae (resolution, R(s) = 6.09, and apparent plate number, N = 2.7-3.3 × 10(5)), were successfully achieved.

Green-Tea and Epigallocatechin-3-Gallate are Bactericidal against Bacillus anthracis

DTIC Science & Technology

2017-06-13

EGCG, catechins such 245 as epigallocatechin and epicatechin gallate are also antibacterial agents. Moreover, the 246 bactericidal activity of green...Sharma A, Gupta S, Sarethy IP, Dang S, Gabrani R. 2012. Green tea extract: possible mechanism 285 and antibacterial activity on skin pathogens. Food...was shown to be responsible for this activity , against 30 both the attenuated B. anthracis ANR and the virulent, encapsulated strain B. anthracis

The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years.

PubMed

Lekota, K E; Hassim, A; Rogers, P; Dekker, E H; Last, R; de Klerk-Lorist, L; van Heerden, H

2018-05-02

Anthrax is a disease with an age old history in Africa caused by the Gram-positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B-clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.

Bacillus Anthracis Comparative Genome Analysis in Support of the Amerithrax Investigation

DTIC Science & Technology

2011-02-02

ability to sporulate . The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of...mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F...a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were

Photoreactivation of Ultraviolet-Irradiated, Plasmid-Bearing and Plasmid-Free Strains of Bacillus anthracis

DTIC Science & Technology

1985-12-19

positive bacterium Bacillus anthracis, is a virulent and highly contagious disease to which most warm-blooded animals, including man, are susceptible... Virulent strains of B. anthracis produce a capsule composed of poly-0-glutamic acid and an exotoxin. The toxin is composed of three proteins identified...as ederma factor (EF), protective antigen (PA), and lethal factor (LF) (17). Anthrax toxin and capsule production are associated with two separate

A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection*

PubMed Central

Vrentas, Catherine E.; Moayeri, Mahtab; Keefer, Andrea B.; Greaney, Allison J.; Tremblay, Jacqueline; O'Mard, Danielle; Leppla, Stephen H.; Shoemaker, Charles B.

2016-01-01

Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LFN, EFN) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax. PMID:27539858

Pathology and pathophysiology of inhalational anthrax in a guinea pig model.

PubMed

Savransky, Vladimir; Sanford, Daniel C; Syar, Emily; Austin, Jamie L; Tordoff, Kevin P; Anderson, Michael S; Stark, Gregory V; Barnewall, Roy E; Briscoe, Crystal M; Lemiale-Biérinx, Laurence; Park, Sukjoon; Ionin, Boris; Skiadopoulos, Mario H

2013-04-01

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Biocidal and Sporicidal Efficacy of Pathoster® 0.35% and Pathoster® 0.50% Against Bacterial Agents in Potential Bioterrorism Use

PubMed Central

Candeliere, Antonio; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

2016-01-01

The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster® 0.35% and Pathoster® 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster® 0.35% was stable until 16±1 days, while Pathoster® 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

Biocidal and Sporicidal Efficacy of Pathoster(®) 0.35% and Pathoster(®) 0.50% Against Bacterial Agents in Potential Bioterrorism Use.

PubMed

Candeliere, Antonio; Campese, Emanuele; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

2016-01-01

The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster(®) 0.35% and Pathoster(®) 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster(®) 0.35% was stable until 16±1 days, while Pathoster(®) 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid.

Modeling low-dose mortality and disease incubation period of inhalational anthrax in the rabbit.

PubMed

Gutting, Bradford W; Marchette, David; Sherwood, Robert; Andrews, George A; Director-Myska, Alison; Channel, Stephen R; Wolfe, Daniel; Berger, Alan E; Mackie, Ryan S; Watson, Brent J; Rukhin, Andrey

2013-07-21

There is a need to advance our ability to conduct credible human risk assessments for inhalational anthrax associated with exposure to a low number of bacteria. Combining animal data with computational models of disease will be central in the low-dose and cross-species extrapolations required in achieving this goal. The objective of the current work was to apply and advance the competing risks (CR) computational model of inhalational anthrax where data was collected from NZW rabbits exposed to aerosols of Ames strain Bacillus anthracis. An initial aim was to parameterize the CR model using high-dose rabbit data and then conduct a low-dose extrapolation. The CR low-dose attack rate was then compared against known low-dose rabbit data as well as the low-dose curve obtained when the entire rabbit dose-response data set was fitted to an exponential dose-response (EDR) model. The CR model predictions demonstrated excellent agreement with actual low-dose rabbit data. We next used a modified CR model (MCR) to examine disease incubation period (the time to reach a fever >40 °C). The MCR model predicted a germination period of 14.5h following exposure to a low spore dose, which was confirmed by monitoring spore germination in the rabbit lung using PCR, and predicted a low-dose disease incubation period in the rabbit between 14.7 and 16.8 days. Overall, the CR and MCR model appeared to describe rabbit inhalational anthrax well. These results are discussed in the context of conducting laboratory studies in other relevant animal models, combining the CR/MCR model with other computation models of inhalational anthrax, and using the resulting information towards extrapolating a low-dose response prediction for man. Published by Elsevier Ltd.

Pathology and Pathophysiology of Inhalational Anthrax in a Guinea Pig Model

PubMed Central

Savransky, Vladimir; Sanford, Daniel C.; Syar, Emily; Austin, Jamie L.; Tordoff, Kevin P.; Anderson, Michael S.; Stark, Gregory V.; Barnewall, Roy E.; Briscoe, Crystal M.; Lemiale-Biérinx, Laurence; Park, Sukjoon; Ionin, Boris

2013-01-01

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's “Animal Rule.” However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 104 spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans. PMID:23357384

A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection.

PubMed

Vrentas, Catherine E; Moayeri, Mahtab; Keefer, Andrea B; Greaney, Allison J; Tremblay, Jacqueline; O'Mard, Danielle; Leppla, Stephen H; Shoemaker, Charles B

2016-10-07

Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LF N , EF N ) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Decontamination of Soil Contaminated with Bacillus anthracis ...

EPA Pesticide Factsheets

Technical Brief This technical summary will provide decontamination personnel rapid access to information on which decontamination approaches are most effective for soils contaminated with B anthracis.

Resistance of bacterial endospores to outer space for planetary protection purposes--experiment PROTECT of the EXPOSE-E mission.

PubMed

Horneck, Gerda; Moeller, Ralf; Cadet, Jean; Douki, Thierry; Mancinelli, Rocco L; Nicholson, Wayne L; Panitz, Corinna; Rabbow, Elke; Rettberg, Petra; Spry, Andrew; Stackebrandt, Erko; Vaishampayan, Parag; Venkateswaran, Kasthuri J

2012-05-01

Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores may withstand certain sterilization procedures as well as the harsh environments of outer space or planetary surfaces. To test their hardiness on a hypothetical mission to Mars, spores of Bacillus subtilis 168 and Bacillus pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space in the experiment PROTECT during the EXPOSE-E mission on board the International Space Station. Mounted as dry layers on spacecraft-qualified aluminum coupons, the "trip to Mars" spores experienced space vacuum, cosmic and extraterrestrial solar radiation, and temperature fluctuations, whereas the "stay on Mars" spores were subjected to a simulated martian environment that included atmospheric pressure and composition, and UV and cosmic radiation. The survival of spores from both assays was determined after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few survivors were recovered from spores exposed in monolayers. Spores in multilayers survived better by several orders of magnitude. All other environmental parameters encountered by the "trip to Mars" or "stay on Mars" spores did little harm to the spores, which showed about 50% survival or more. The data demonstrate the high chance of survival of spores on a Mars mission, if protected against solar irradiation. These results will have implications for planetary protection considerations.

Hollow-Fiber Pharmacodynamic Studies and Mathematical Modeling To Predict the Efficacy of Amoxicillin for Anthrax Postexposure Prophylaxis in Pregnant Women and Children

PubMed Central

VanScoy, Brian; Liu, Weiguo; Kulawy, Robert; Drusano, G. L.

2013-01-01

Amoxicillin is considered an option for postexposure prophylaxis of Bacillus anthracis in pregnant and postpartum women who are breastfeeding and in children because of the potential toxicities of ciprofloxacin and doxycycline to the fetus and child. The amoxicillin regimen that effectively kills B. anthracis and prevents resistance is unknown. Fourteen-day dose range and dose fractionation studies were conducted in in vitro pharmacodynamic models to identify the exposure intensity and pharmacodynamic index of amoxicillin that are linked with optimized killing of B. anthracis and resistance prevention. Studies with dicloxacillin, a drug resistant to B. anthracis beta-lactamase, evaluated the role of beta-lactamase production in the pharmacodynamic indices for B. anthracis killing and resistance prevention. Dose fractionation studies showed that trough/MIC and not time above MIC was the index for amoxicillin that was linked to successful outcome through resistance prevention. Failure of amoxicillin regimens was due to inducible or stable high level expression of beta-lactamases. Studies with dicloxacillin demonstrated that a time above MIC of ≥94% was linked with treatment success when B. anthracis beta-lactamase activity was negated. Recursive partitioning analysis showed that amoxicillin regimens that produced peak concentrations of <10.99 μg/ml and troughs of >1.75 μg/ml provided a 100% success rate. Other amoxicillin peak and trough values produced success rates of 28 to 67%. For postpartum and pregnant women and children, Monte Carlo simulations predicted success rates for amoxicillin at 1 g every 8 h (q8h) of 53, 33, and 44% (30 mg/kg q8h), respectively. We conclude that amoxicillin is suboptimal for postexposure prophylaxis of B. anthracis in pregnant and postpartum women and in children. PMID:24041894

An evaluation of suspicious powder screening tools for first responders.

PubMed

Poore, Carrie; Clark, Paul; Emanuel, Peter A

2009-12-30

Field screening tools are required which would allow first responders to quickly ascertain if a suspicious powder poses a potential threat necessitating additional testing for biological pathogens such as Bacillus anthracis. In this study, three commercially available generic screening technologies were evaluated for the effectiveness to accurately differentiate between a hoax powder and a true biological threat. The BioCheck Kit was able to detect the following biological agents 1 x 10(8)CFU of B. anthracis Sterne (washed 4 times), 1x10(7)CFU of B. anthracis DeltaSterne (washed 2 times), 1 x 10(7)CFU of Yersinia pestis A1122, and 100 microg of ricin. The Prime Alert kit was able to detect 2 x 10(10)CFU of B. anthracis DeltaSterne 4x, 1 x 10(9)CFU of B. anthracis DeltaSterne 2x, and 1 x 10(8)CFU of Y. pestis A1122. The Prime Alert kit was not able to detect ricin. The Profile-1 kit was able to detect 1 x 10(4)CFU of B. anthracis DeltaSterne 4x and B. anthracis DeltaSterne 2x, and 1 x 10(6)CFU of Y. pestis A1122. The Profile-1 kit was not able to detect ricin. All of the kits showed positive results for powders containing components specifically targeted by the particular technology being used. Each technology assessed in this evaluation employs a different mechanism for the detection of biological materials and it is important that first responders are aware of the strengths and the limitations of each system so that they can effectively employ the technology to protect the homeland.

Transponson Tn916 Mutagenesis in Bacillus anthracis,

DTIC Science & Technology

1987-11-10

Tngla, is described. Tng1a was transferred from Streptococcus 1aJaji strain DS16C1 to f. a VNR-1 by conjugation in a standard filter mating procedure...transposon, Tn916, mutagenesis, Bacillus, anthracis, subtilis. , Streptococcus , faecalis, aro 2.AUSrN ACT (Cautious no reverse efho if nece.at7r sd ideratfy...transferred from Streptococcus : faecalis strain DS16CI to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline

Western Blot Analysis of the Exotoxin Components From Bacillus anthracis Separated by Isoelectric Focusing Gel Electrophoresis

DTIC Science & Technology

2004-02-14

measured by using in vitro cell-based assays. This study provides another method of characterizing various isolates of B . anthracis by determining the...isoelectric points of the exotoxin components and may be useful in the development of protective vaccines against B . anthracis infection. 15. SUBJECT...LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES 11 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b . ABSTRACT unclassified c. THIS PAGE

Differences in Susceptibility of Inbred Mice to Bacillus anthracis

DTIC Science & Technology

1985-04-26

dilutions of the mixture were prepared and injected into A/J and CBA/J mice via the tail vein, as described by Ezzell et al. (9). Five mice per strain were...xylazine (Rompun, Miles Laboratories, Shawnee, Kansas) in 50 pl, and were dissected iwnmediately. Gross pathological changes were noted, heart blood and...anthracis; a histopathological study of skin lesions produced by B. anthracis in susceptible and resistant animal species. J. Infect. Dis. 80:1-13. 9. Ezzell

Rapid PCR Assays That Specifically Identify Anthrax and Anthrax Surrogate Chromosomal Signatures

DTIC Science & Technology

2002-08-30

The genetic variation among a set of 175 full-length sspE DNA sequences obtained from representative members of the B. anthracis clade have been...examined. Thirty-six sspE genotypes and seventeen protein phylotypes were identified among the B. cereus, B. thuringiensis, B. anthracis and B. mycoides...the sspE DNA sequence data sets suggests that the B. anthracis dade is more phylogenetically complex than has been inferred by traditional taxonomic methods.

Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis

PubMed Central

Barro, Alassane S.; Fegan, Mark; Moloney, Barbara; Porter, Kelly; Muller, Janine; Warner, Simone; Blackburn, Jason K.

2016-01-01

The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP), historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies. PMID:27280981

Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis.

PubMed

Barro, Alassane S; Fegan, Mark; Moloney, Barbara; Porter, Kelly; Muller, Janine; Warner, Simone; Blackburn, Jason K

2016-06-01

The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP), historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies.

Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat

PubMed Central

Weber-Dąbrowska, Beata; Borysowski, Jan; Górski, Andrzej

2014-01-01

Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat. PMID:25247187

Genetic and Physiological Studies of Bacillus anthracis Related to Development of an Improved Vaccine

DTIC Science & Technology

1987-07-01

nontransformable Bacillus species such as B. anthracis. Our results suggest that plasmid pLS20 of Bacillus subtilis ( natto ), which promotes transfer of the...mobilizing pBC16, pLS20 mediates transfer of the B. subtills ( natto ) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. To facilitate direct...and (v) transformation of B. cereus and B. anthracis with plasmid DNA. The 55-kb plasmid, pLS20, of Bacillus subtilis ( natto ) 3335 promotes tr msfer

Genetic and Physiological Studies of Bacillus Anthracis Related to Development of An Improved Vaccine

DTIC Science & Technology

1988-07-01

to replicate at all in B. anthracis. Such mutants al currently being tested as vehicles for transposon mutagenesis in B. anthracis. B. subtilis ( natto ...subtilis ( natto ) 3335 harbors a plasmid, pLS20, which encodes functions responsible for conjugal transfer of plasmids among genetically re- lated and...Cultures were grown for 16 hours in BHI broth supplemented with 0.1% glycerol, o- in LG broth for B. subtilis natto . After cell pellets were suspended in

Geographical Distribution of Genotypic and Phenotypic Markers Among Bacillus Anthracis Isolates and Related Species by Historical Movement and Horizontal Transfer

DTIC Science & Technology

2009-01-09

LOPEZ P., ESPINOSA M., PIECHOWSAK M., SHUGAR D., WARREN R.: Uptake and fate of ΦW-14 DNA in competent Bacillus subtilis . J.Bacteriol. 149, 595–605...Among Bacillus anthracis Isolates and Related Species by Historical Movement and Horizontal Transfer J.L. KIELa, J.E. PARKERa, E.A. HOLWITTa, R.P...The geographical distribution of Bacillus anthracis strains and isolates bearing some of the same genetic markers as the Amerithrax Ames isolate was

Nosocomial Infection of Serratia marcescens May Induce a Protective Effect of Monkeys Exposed to Bacillus anthracis

DTIC Science & Technology

2008-01-01

B. anthracis or a detectable level of protective antigen in the bloodstream. It appears that the presence of S . marcescens may have induced a "Coley’s...Available online 6 June 2008KEYWORDS Inhalation anthrax; Innate immunity; B. anthracis; S . marcescens ; African green monkey* Corresponding author. Tel.: þ1 30...had S . marcescens contam- ination in the catheter; indicated by pure colonies grown from the blood. None of these AGMs showed clinical signs of illness

Resistance of Bacterial Endospores to Outer Space for Planetary Protection Purposes—Experiment PROTECT of the EXPOSE-E Mission

PubMed Central

Moeller, Ralf; Cadet, Jean; Douki, Thierry; Mancinelli, Rocco L.; Nicholson, Wayne L.; Panitz, Corinna; Rabbow, Elke; Rettberg, Petra; Spry, Andrew; Stackebrandt, Erko; Vaishampayan, Parag; Venkateswaran, Kasthuri J.

2012-01-01

Abstract Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores may withstand certain sterilization procedures as well as the harsh environments of outer space or planetary surfaces. To test their hardiness on a hypothetical mission to Mars, spores of Bacillus subtilis 168 and Bacillus pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space in the experiment PROTECT during the EXPOSE-E mission on board the International Space Station. Mounted as dry layers on spacecraft-qualified aluminum coupons, the “trip to Mars” spores experienced space vacuum, cosmic and extraterrestrial solar radiation, and temperature fluctuations, whereas the “stay on Mars” spores were subjected to a simulated martian environment that included atmospheric pressure and composition, and UV and cosmic radiation. The survival of spores from both assays was determined after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few survivors were recovered from spores exposed in monolayers. Spores in multilayers survived better by several orders of magnitude. All other environmental parameters encountered by the “trip to Mars” or “stay on Mars” spores did little harm to the spores, which showed about 50% survival or more. The data demonstrate the high chance of survival of spores on a Mars mission, if protected against solar irradiation. These results will have implications for planetary protection considerations. Key Words: Planetary protection—Bacterial spores—Space experiment—Simulated Mars mission. Astrobiology 12, 445–456. PMID:22680691

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores.

PubMed

Mazza, Paola; Noens, Elke E; Schirner, Kathrin; Grantcharova, Nina; Mommaas, A Mieke; Koerten, Henk K; Muth, Günther; Flärdh, Klas; van Wezel, Gilles P; Wohlleben, Wolfgang

2006-05-01

MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.

76 FR 53480 - Prospective Grant of Exclusive License: Conjugate Vaccines Against B. anthracis

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-26

.... anthracis Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF)'', U.S. Patent Application... catalytic proteins known as ``lethal factor'' (LF) and ``edema factor'' (EF). Although production of an...

Detecting anthrax in the palm of your hand: applications of a smartphone microscope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erikson, Rebecca L.; Hutchison, Janine R.

Bacillus anthracis is a bacterial pathogen that causes the disease anthrax. In 2001, B. anthracis was used in a bioterrorism attack in the United States that resulted in 22 individuals becoming infected, 5 of whom died as a result of this attack. A great deal of attention has been dedicated to responding to bioterrorism events to reduce the potential loss of lives. One such area of research has focused on the development of new technologies to detect and respond to the intentional release of bacterial pathogens such as B. anthracis.

EPA/CDC Interim Clearance Strategy for Environments Contaminated with Anthrax

EPA Pesticide Factsheets

Strategy for public health and environmental Federal responders to aid Incident Command/Unified Command (IC/UC) in clearing a building or an outdoor environment after an incident involving contamination with Bacillus anthracis (B. anthracis)

Composite Sampling of a Bacillus anthracis Surrogate with ...

EPA Pesticide Factsheets

Journal Article A series of experiments were conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a stainless steel surface.

Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

DOEpatents

Bavykin, Sergei G.; Mirzabekova, legal representative, Natalia V.; Mirzabekov, deceased, Andrei D.

2007-12-04

The present invention relates to methods and compositions for using nucleotide sequence variations of 16S and 23S rRNA within the B. cereus group to discriminate a highly infectious bacterium B. anthracis from closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations and discriminate B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed samples, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

PubMed

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

DOEpatents

Bavykin, Sergei G.; Mirzabekov, Andrei D.

2007-10-30

The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

PubMed Central

Marston, Chung K.; Bhullar, Vinod; Baker, Daniel; Rahman, Mahmudur; Hossain, M. Jahangir; Chakraborty, Apurba; Khan, Salah Uddin; Hoffmaster, Alex R.

2012-01-01

The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at ≥7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions. PMID:23035192

Ultrastructure of developing ascospores in Sordaria brevicollis.

PubMed

Hackett, C J; Chen, K C

1976-05-01

The ultrastructure of ascospore wall formation in the pyrenomycete Sordaria brevicollis was studied in developing asci at progressive time intervals. From early spore delimitation through final stage of maturation, the wall of the ascospore differentiated into four composite layers, the periascosporium the delineation ascosporium, the subascosproium, and the endoascosproium, While ascospores were at the hyaline stage of development,they possessed only the periascosporium and delineation ascosporium as their wall components. At about 7 to 8 days from the initiation of the cross, the spores developed a yellow color, and this coloration was always associated with the elaboration of the subascorsporium just internal to the ascosporium. Asthe spores continued to progressively darken in color, the subascosporium was seen to increase in complexity, electron density, and thickness. Soon after the formation of the subascosporium, the endoascosporium began to develop de novo and was, therefore, the last wall layer formed as the spore approached maturity.

In vitro production of Clostridium difficile spores for use in the efficacy evaluation of disinfectants: a precollaborative investigation.

PubMed

Hasan, Jafrul A; Japal, Knoxley M; Christensen, Erick R; Samalot-Freire, Luisa C

2011-01-01

Clostridium difficile is a strict anaerobic spore-forming bacterium, and an increasingly common nosocomial pathogen. The U.S. Environmental Protection Agency (EPA) is responsible for the registration of disinfectants, including products designed to treat environmental surfaces contaminated with spores of C. difficile. Product efficacy data are required for registration; however, there is a lack of methodology for generating high-quality spore suspensions for evaluating product performance. As such, a study was carried out to select a suitable C. difficile strain and to develop a stand-alone method to prepare a spore suspension that meets specific criteria necessary for quantitative testing of disinfectants. The criteria are: (1) a spore titer of > 8 log10/mL, (2) > or = 90% spores to vegetative cells, and (3) resistance of spores (determined by viability) to 2.5 M hydrochloric acid (HCl). Several strains of C. difficile (toxigenic and nontoxigenic) were grown on various media (solid and liquid) for varying lengths of time to determine the best combination of incubation conditions and media to optimize spore production and quality. Once the spore production procedure was optimized, a toxigenic strain of C. difficile [American Type Culture Collection (ATCC) 43598] was selected for use in trials to verify repeatability from one production run to the next. The spore suspension was initiated by spreading vegetative cells of C. difficile (ATCC 43598) on CDC anaerobic 5% sheep blood agar plates and incubating for 7-10 days at 36 +/- 1 degrees C under anaerobic conditions. Spores were harvested when > or = 90% of the cells converted to spores as determined by observation using phase-contrast microscopy. The spores were washed three times with saline-Tween-80, resuspended in cold deionized water, heated to 70 degrees C for 10 min, evaluated microscopically for quality, and enumerated on cycloserine-cefoxitin-fructose agar containing horse blood and taurocholate. The spore suspension was used to inoculate brushed stainless steel carriers (1 cm in diameter) with and without a soil load in accordance with the Standard Quantitative Carrier Disk Test Method (ASTM E-2197-02) to determine carrier load. Once it was determined that > 6 log10 spores/carrier could be recovered, spores were evaluated for resistance to HCI. The sporulation method presented in this report is simple and repeatable and results in spore suspension of high titer (> 8 log10/mL) and quality (> or = 90% spores to vegetative cells) that met acid resistance criteria (spores were resistant to 2.5 M HCI for 10 min). In addition, recovery from brushed stainless steel carriers with and without soil load was > 6 log10 spores/carrier. A 6 log10 performance standard was set forth in the EPA's interim guidance for generating data to support a label claim for effectiveness against C. difficile spores on hard, nonporous surfaces. This precollaborative investigation successfully demonstrated the use of a methodology for in vitro production of C. difficile spores (ATCC 43598) necessary for conducting efficacy tests. A proposal will be submitted to the AOAC INTERNATIONAL Methods Committee on Antimicrobial Efficacy Testing for a collaborative study; see Appendix.

Comparisons of the humoral and cellular immunity induced by live A16R attenuated spore and AVA-like anthrax vaccine in mice.

PubMed

Lv, Jin; Zhang, Ying-Ying; Lu, Xun; Zhang, Hao; Wei, Lin; Gao, Jun; Hu, Bin; Hu, Wen-Wei; Hu, Dun-Zhong; Jia, Na; Feng, Xin

2017-03-01

The live attenuated anthrax vaccine and anthrax vaccine adsorbed (AVA) are two main types of anthrax vaccines currently used in human. However, the immunoprotective mechanisms are not fully understood. In this study, we compared humoral and cellular immunity induced by live A16R spore vaccine and A16R strain derived AVA-like vaccine in mice peripheral blood, spleen and bone marrow. Both A16R spores and AVA-like vaccines induced a sustained IgG antibody response with IgG1/IgG2b subtype dominance. However, A16R spores vaccine induced higher titer of IgG2a compared with AVA-like vaccine, indicating a stronger Th1 response to A16R spores. Using antigen-specific ELISpot assay, we observed a significant response of ASCs (antibody secreting cells) and IL4-CSCs (cytokine secreting cells) in mice. Specially, there was a positive correlation between the frequencies of antigen specific ASCs and IL4-CSCs in bone marrow derived cells, either by A16R spore or AVA-like vaccine vaccination. Moreover, we also found A16R spore vaccine, not AVA-like vaccine, could induce sustained frequency of IFN-γ-CSCs in bone marrow derived cells. Collectively, both the vaccines induced a mixed Th1/Th2 response with Th2 dominance in mice and A16R spore vaccine might provide a more comprehensive protection because of humoral and cellular immunity induced in bone marrow. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Multichannel waveguides for the simultaneous detection of disease biomarkers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukundan, Harshini; Price, Dominique Z; Grace, Wynne K

2009-01-01

The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor that has previously been used for the detection of biomarkers associated with diseases such as tuberculosis, breast cancer, anthrax and influenza in complex biological samples (e.g., serum and urine). However, no single biomarker can accurately predict disease. To address this issue, we developed a multiplex assay for the detection of components of the Bacillus anthracis lethal toxin on single mode planar optical waveguides with tunable quantum dots as the fluorescence reporter. This limited ability to multiplex is still insufficient for accurate detection of disease ormore » for monitoring prognosis. In this manuscript, we demonstrate for the first time, the design, fabrication and successful evaluation of a multichannel planar optical waveguide for the simultaneous detection of at least three unknown samples in quadruplicate. We demonstrate the simultaneous, rapid (30 min), quantitative (with internal standard) and sensitive (limit of detection of 1 pM) detection of protective antigen and lethal factor of Bacillus anthracis in complex biological samples (serum) using specific monoclonal antibodies labeled with quantum dots as the fluorescence reporter.« less

Inhalational anthrax (Ames aerosol) in naïve and vaccinated New Zealand rabbits: characterizing the spread of bacteria from lung deposition to bacteremia

PubMed Central

Gutting, Bradford W.; Nichols, Tonya L.; Channel, Stephen R.; Gearhart, Jeffery M.; Andrews, George A.; Berger, Alan E.; Mackie, Ryan S.; Watson, Brent J.; Taft, Sarah C.; Overheim, Katie A.; Sherwood, Robert L.

2012-01-01

There is a need to better understand inhalational anthrax in relevant animal models. This understanding could aid risk assessment, help define therapeutic windows, and provide a better understanding of disease. The aim here was to characterize and quantify bacterial deposition and dissemination in rabbits following exposure to single high aerosol dose (> 100 LD50) of Bacillus anthracis (Ames) spores immediately following exposure through 36 h. The primary goal of collecting the data was to support investigators in developing computational models of inhalational anthrax disease. Rabbits were vaccinated prior to exposure with the human vaccine (Anthrax Vaccine Adsorbed, AVA) or were sham-vaccinated, and were then exposed in pairs (one sham and one AVA) so disease kinetics could be characterized in equally-dosed hosts where one group is fully protected and is able to clear the infection (AVA-vaccinated), while the other is susceptible to disease, in which case the bacteria are able to escape containment and replicate uncontrolled (sham-vaccinated rabbits). Between 4–5% of the presented aerosol dose was retained in the lung of sham- and AVA-vaccinated rabbits as measured by dilution plate analysis of homogenized lung tissue or bronchoalveolar lavage (BAL) fluid. After 6 and 36 h, >80% and >96%, respectively, of the deposited spores were no longer detected in BAL, with no detectable difference between sham- or AVA-vaccinated rabbits. Thereafter, differences between the two groups became noticeable. In sham-vaccinated rabbits the bacteria were detected in the tracheobronchial lymph nodes (TBLN) 12 h post-exposure and in the circulation at 24 h, a time point which was also associated with dramatic increases in vegetative CFU in the lung tissue of some animals. In all sham-vaccinated rabbits, bacteria increased in both TBLN and blood through 36 h at which point in time some rabbits succumbed to disease. In contrast, AVA-vaccinated rabbits showed small numbers of CFU in TBLN between 24 and 36 h post-exposure with small numbers of bacteria in the circulation only at 24 h post-exposure. These results characterize and quantify disease progression in naïve rabbits following aerosol administration of Ames spores which may be useful in a number of different research applications, including developing quantitative models of infection for use in human inhalational anthrax risk assessment. PMID:22919678

An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics.

PubMed

Okinaka, Richard T; Price, Erin P; Wolken, Spenser R; Gruendike, Jeffrey M; Chung, Wai Kwan; Pearson, Talima; Xie, Gary; Munk, Chris; Hill, Karen K; Challacombe, Jean; Ivins, Bruce E; Schupp, James M; Beckstrom-Sternberg, Stephen M; Friedlander, Arthur; Keim, Paul

2011-09-30

An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.

An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics

PubMed Central

2011-01-01

Background An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. Results We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. Conclusions We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence. PMID:21962024

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

PubMed Central

Braun, Peter; Grass, Gregor; Aceti, Angela; Serrecchia, Luigina; Affuso, Alessia; Marino, Leonardo; Grimaldi, Stefania; Pagano, Stefania; Hanczaruk, Matthias; Georgi, Enrico; Northoff, Bernd; Schöler, Anne; Schloter, Michael; Antwerpen, Markus; Fasanella, Antonio

2015-01-01

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis. PMID:26266934

Whole-Genome Sequences of Variants of Bacillus anthracis Sterne and Their Toxin Gene Deletion Mutants

PubMed Central

Staab, A.; Plaut, R. D.; Pratt, C.; Lovett, S. P.; Wiley, M. R.; Biggs, T. D.; Bernhards, R. C.; Beck, L. C.; Palacios, G. F.; Stibitz, S.; Jones, K. L.; Goodwin, B. G.; Smith, M. A.

2017-01-01

ABSTRACT Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives. PMID:29122874

Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation†

PubMed Central

Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

2006-01-01

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis. PMID:16385039

Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation.

PubMed

Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

2006-01-01

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

Assembly and Function of the Bacillus anthracis S-Layer.

PubMed

Missiakas, Dominique; Schneewind, Olaf

2017-09-08

Bacillus anthracis, the anthrax agent, is a member of the Bacillus cereus sensu lato group, which includes invasive pathogens of mammals or insects as well as nonpathogenic environmental strains. The genes for anthrax pathogenesis are located on two large virulence plasmids. Similar virulence plasmids have been acquired by other B. cereus strains and enable the pathogenesis of anthrax-like diseases. Among the virulence factors of B. anthracis is the S-layer-associated protein BslA, which endows bacilli with invasive attributes for mammalian hosts. BslA surface display and function are dependent on the bacterial S-layer, whose constituents assemble by binding to the secondary cell wall polysaccharide (SCWP) via S-layer homology (SLH) domains. B. anthracis and other pathogenic B. cereus isolates harbor genes for the secretion of S-layer proteins, for S-layer assembly, and for synthesis of the SCWP. We review here recent insights into the assembly and function of the S-layer and the SCWP.

Over-expression, purification, and confirmation of Bacillus anthracis transcriptional regulator NprR

PubMed Central

Rice, Amy J.; Woo, Jerry K.; Khan, Attiya; Szypulinski, Michael Z.; Johnson, Michael E.; Lee, Hyunwoo; Lee, Hyun

2016-01-01

Quorum sensing (QS) has been recognized as an important biological phenomenon in which bacterial cells communicate and coordinate their gene expression and cellular processes with respect to population density. Bacillus anthracis is the etiological agent of fatal pulmonary anthrax infections, and the NprR/NprX QS system may be involved in its pathogenesis. NprR, renamed as aqsR for anthrax quorum sensing Regulator, is a transcriptional regulator that may control the expression of genes required for proliferation and survival. Currently, there is no protocol reported to over-express and purify B. anthracis AqsR. In this study, we describe cloning, purification, and confirmation of functional full-length B. anthracis AqsR protein. The AqsR gene was cloned into the pQE-30 vector with an HRV 3C protease recognition site between AqsR and the N-terminal His6-tag in order to yield near native AqsR after the His-tag cleavage, leaving only two additional amino acid residues at the N-terminus. PMID:26344899