Tonya Nichols

Inhalational anthrax(Ames aerosol)in naïve and vaccinated New Zealand rabbits: characterizing the spread of bacteria from lung deposition to bacteremia

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.

Recent literature review of soil processing methods for recovery of Bacillus anthracis spores

Identifying virulent Bacillus anthracis within soil is a difficult task due to the number and diversity of other organisms and impeding chemical constituents within soil. Regardless of the detection assay, the initial sample must be processed efficiently to ensure that debris, chemical components, and biological impurities do not obstruct downstream analysis. Soil sample processing protocols can be divided into two general types: indirect and direct. There are two requirements for successful indirect isolation of B. anthracis from soil samples: dissociate the spores from the soil particles and physically separate the free spores from the soil particles. Adding an aqueous carrier medium to a soil sample creates a sample slurry for easier manipulation. Centrifugation, high specific gravity separation, immunomagnetic separation, filtration, and settling have been used to physically separate spores from soil. Direct processing utilizes a soil sample without first separating the spores from the bulk sample and falls under two principal types: culturing on B. anthracis selective agar and bulk DNA extraction. Direct and indirect processing steps each have associated advantages and disadvantages. The objective of this review was to consolidate information acquired from previous research, focusing primarily on data gleaned in the last decade, on the processing of soils contaminated with B. anthracis. As shown in this review, an optimized soil-processing protocol with a known recovery rate and associated confidence intervals is needed. A reliable processing protocol would allow for multiple investigators and laboratories to produce high-quality, uniform results in the event of a B. anthracis release.

Achieving consistent multiple daily low-dose Bacillus anthracis spore inhalation exposures in the rabbit model

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.

Observations on the Migration of Bacillus Spores Outside a Contaminated Facility During a Decontamination Efficacy Study

The fate and transport of Bacillus anthracis spores in indoor and outdoor environments is not well understood. The Bio-Response Operational Testing and Evaluation exercise evaluated decontamination technologies in a twostory building experimentally contaminated with Bacillus atrophaeus subspecies globigii spores. The Bio-Response Operational Testing and Evaluation project provided a means to evaluate the potential for the spores dispersed inside the building to migrate to the outside as well as to investigate a new method for processing soils contaminated with Bacillus spores. Duplicate sterile sand samples were placed within the tent covering the building, but outside the building itself, near entrances, exits, and high-traffic areas to assess migration and deposition of newly disseminated spores. The sand samples were utilized during three stages of the decontamination study: before spore dissemination, after spore dissemination, and after decontamination of the building. In addition, two sets of sand samples placed within the building provided positive controls. Results from two different building decontamination approaches were studied. Results were tabulated as presence or absence rather than as a quantitative figure. There was no significant association among positive samples and the location of the samples around the building. There was a significant association between the different stages of each decontamination study and the number of detectable samples. The results of this study demonstrate the potential for spores to migrate out of a contaminated building and the importance of considering migration when assessing the scope of a contamination incident

Operational evaluation of the rapid viability PCR method for post-decontamination clearance sampling.

The Rapid Viability Polymerase Chain Reaction (RV-PCR) method was evaluated during the Bio-Response Operational Testing and Evaluation (BOTE), an interagency project to evaluate field-level facility biological remediation, using leading decontamination technologies. The tests were performed using an intentional release (aerosolization) of spores of Bacillus atrophaeus subspecies globigii (BG), as a surrogate for Bacillus anthracis, the etiologic agent for anthrax. Three decontamination methods were assessed including fumigation with vaporized hydrogen peroxide (VHP), fumigation with chlorine dioxide (CD), and a surface treatment process using pH-adjusted bleach.The RV-PCR method was developed to rapidly detect live B. anthracis spores during a bioterrorism event. The method uses a change in realtime PCR response before and after a nine hour incubation step, to determine the presence of viable bacterial spores in the sample; the method was recently verified for air filter, wipe and water samples at the 10-spore level for B. anthracis Ames spores, and was also developed for swab, sponge-stick, and vacuum sock/filter samples. In the method, high throughput sample processing is combined with PCR-based analysis before and after a rapid culture step to speed viability determination, especially for complex surface and environmental samples that present challenges to current culture-based methods. In the BOTE project, a total of 159 surface wipe samples from post-decontamination events were analyzed by splitting the suspension after spore recovery into two equal parts, with one part analyzed by RV-PCR and the other part by culture after concentrating to the same volume. In the BOTE project, the RV-PCR method provided rapid results for post-decontamination samples that were 98% (156/159 samples) consistent with results from culture analysis. The percentage agreement was noteworthy, given the large number of samples containing low spore levels. For the Post-VHP, Post-Bleach, and Post-CD event samples, the percentage agreement was 93% (41/44 samples), 100% (47/47 samples), and 100% (68/68 samples), respectively. The RV-PCR method performed well for the surrogate BG spores exposed to decontaminants at real-world application levels, and with wipe samples containing background debris and indigenous microbial populations.

Considerations for estimating microbial environmental data concentrations collected from a field setting.

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.

Analysis of environmental contamination resulting from catastrophic incidents: Part 1. Building and sustaining capacity in laboratory networks

Catastrophic incidents, such as natural disasters, terrorist attacks, and industrial accidents, can occur suddenly and have high impact. However, they often occur at such a low frequency and in unpredictable locations that planning for the management of the consequences of a catastrophe can be difficult. For those catastrophes that result in the release of contaminants, the ability to analyze environmental samples is critical and contributes to the resilience of affected communities. Analyses of environmental samples are needed to make appropriate decisions about the course of action to restore the area affected by the contamination. Environmental samples range from soil, water, and air to vegetation, building materials, and debris. In addition, processes used to decontaminate any of these matrices may also generate wastewater and other materials that require analyses to determine the best course for proper disposal. This paper summarizes activities and programs the United States Environmental Protection Agency (USEPA) has implemented to ensure capability and capacity for the analysis of contaminated environmental samples following catastrophic incidents. USEPAs focus has been on building capability for a wide variety of contaminant classes and on ensuring national laboratory capacity for potential surges in the numbers of samples that could quickly exhaust the resources of local communities. USEPAs efforts have been designed to ensure a strong and resilient laboratory infrastructure in the United States to support communities as they respond to contamination incidents of any magnitude. The efforts include not only addressing technical issues related to the best-available methods for chemical, biological, and radiological contaminants, but also include addressing the challenges of coordination and administration of an efficient and effective response. Laboratory networks designed for responding to large scale contamination incidents can be sustained by applying their resources during incidents of lesser significance, for special projects, and for routine surveillance and monitoring as part of ongoing activities of the environmental laboratory community.

Analysis of environmental contamination resulting from catastrophic incidents: part 2. Building laboratory capability by selecting and developing analytical methodologies.

Catastrophic incidents can generate a large number of samples of analytically diverse types, including forensic, clinical, environmental, food, and others. Environmental samples include water, wastewater, soil, air, urban building and infrastructure materials, and surface residue. Such samples may arise not only from contamination from the incident but also from the multitude of activities surrounding the response to the incident, including decontamination. This document summarizes a range of activities to help build laboratory capability in preparation for sample analysis following a catastrophic incident, including selection and development of fit-for-purpose analytical methods for chemical, biological, and radiological contaminants. Fit-for-purpose methods are those which have been selected to meet project specific data quality objectives. For example, methods could be fit for screening contamination in the early phases of investigation of contamination incidents because they are rapid and easily implemented, but those same methods may not be fit for the purpose of remediating the environment to acceptable levels when a more sensitive method is required. While the exact data quality objectives defining fitness-for-purpose can vary with each incident, a governing principle of the method selection and development process for environmental remediation and recovery is based on achieving high throughput while maintaining high quality analytical results. This paper illustrates the result of applying this principle, in the form of a compendium of analytical methods for contaminants of interest. The compendium is based on experience with actual incidents, where appropriate and available. This paper also discusses efforts aimed at adaptation of existing methods to increase fitness-for-purpose and development of innovative methods when necessary. The contaminants of interest are primarily those potentially released through catastrophes resulting from malicious activity. However, the same techniques discussed could also have application to catastrophes resulting from other incidents, such as natural disasters or industrial accidents. Further, the high sample throughput enabled by the techniques discussed could be employed for conventional environmental studies and compliance monitoring, potentially decreasing costs and/or increasing the quantity of data available to decision-makers.

Evaluation of exposure to Brevundimonas diminuta and Pseudomonas aeruginosa during showering

This study experimentally assessed bacterial water-to-air partitioning coefficients resulting from showerhead aerosolization of water contaminated with Brevundimonas diminuta or Pseudomonas aeruginosa, and estimated human exposure through inhalation. Dechlorinated tap water was spiked with two cell densities (109 and 1010 CFU l-1) and cycled at three temperatures (10, 25, and 37 or 40ºC) through a full-scale shower system. For reproducibility, spiked water concentrations were intentionally higher than found in natural environments. Three types of samplers measured size distribution and viable concentrations throughout the system. Results indicate low levels of respirable bioaerosols were generated. The ratio of bacterial contaminant that was effectively aerosolized (bacterial water-to-air partitioning coefficient, PC bwa ) was low - averaging 1.13×10-5 L m-3 for B. diminuta and 8.31×10-6 L m-3 for P. aeruginosa. However, the respirable fraction of aerosolized organisms was high, averaging above 94% (in shower) and above 99% (downstream) for both organisms. This study found no significant difference in bioaerosol load for a forward facing versus reverse facing individual. Further, for the average hot shower (33-43°C) the total number of respirable bioaerosols is higher, but the observed culturability of those aerosolized cells is lower when compared to lower temperatures. Bacterial water to air partitioning coefficients were calculated to predict microbial air concentration and these empirical parameters may be used for assessing inhalation as a route of exposure to pathogens in contaminated waters.

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

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 14spores/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.