Physiological Responses to Multiple Low-Doses of Bacillus anthracis Spores in the Rabbit Model of Inhalation Anthrax.

Bacillus anthracis spores that are re-aerosolized from surface deposits after initial contamination present significant health risks for personnel involved in decontamination. To model repeated exposure to low dose B. anthracis spores, three groups of seven rabbits were challenged with multiple low-doses of B. anthracis spores 5 days a week for 3 weeks. Mortality, body temperature, heart and respiration rates, hematology, C-reactive protein, bacteremia, and serum protective antigen were monitored for 21 days post-exposure after the last of multiple doses. All rabbits exposed to a mean daily dose of 2.91 × 102 colony forming units (CFU) survived and showed minimal physiological changes attributable to exposure. One of seven rabbits receiving a mean daily dose of 1.22 × 103 CFU died and four of seven receiving a mean daily dose of 1.17 × 104 CFU died. The LD50 was calculated to be 8.1 × 103 CFU of accumulated dose. Rabbits that succumbed to the higher dose exhibited bacteremia and increases above baseline in heart rate, respiration rate, and body temperature. Two rabbits in the mean daily dose group of 1.17 × 104 CFU exhibited clinical signs of inhalation anthrax yet survived. This study provides a description of lethality, pathophysiology, and pathology in a controlled multiple low-dose inhalation exposure study of B. anthracis in the rabbit model. The data suggest that the accumulated dose is important in survival outcome and that a subset of rabbits may show clinical signs of disease but fully recover without therapeutic intervention.

Physiological Responses to a Single Low-Dose of Bacillus anthracis Spores in the Rabbit Model of Inhalational Anthrax.

Credible dose-response relationships are needed to more accurately assess the risk posed by exposure to low-level Bacillus anthracis contamination during or following a release. To begin to fill this knowledge gap, New Zealand White rabbits were implanted with D70-PCT telemetry transmitters and subsequently aerosol challenged with average inhaled doses of 2.86 x 102 to 2.75 x 105 colony forming units (CFU) of B. anthracis spores. Rabbits exposed to a single inhaled dose at or above 2.54 × 104 CFU succumbed with dose-dependent time to death. Death was associated with increases above baseline in heart rate, respiration rate, and body temperature and all rabbits that died exhibited bacteremia at some point prior to death. Rabbits that inhaled doses of 2.06 × 103 CFU or lower survived to the end of the study and showed no or minimal adverse changes in the measured physiological responses in response to the challenge. Moreover, no bacteremia nor toxemia were observed in rabbits that survived to the end of the study. Overall, the data indicate that challenge doses of B. anthracis below the level sufficient to establish systemic infection do not produce observable physiological responses; however, doses that triggered a response resulted in death.

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.

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.

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.

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 10(6) 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 × 10(2), 1 × 10(3), 1 × 10(4), and 1 × 10(5) CFU were used. In the multiple daily exposure experiment, rabbits were exposed multiple days to targeted inhaled doses of 1 × 10(2), 1 × 10(3), and 1 × 10(4) 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.

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 LD(50)) 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.