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.

Modeling Rabbit Responses to Single and Multiple Aerosol Exposures of Bacillus anthracis Spores.

Survival models are developed to predict response and time-to-response for mortality in rabbits following exposures to single or multiple aerosol doses of Bacillus anthracis spores. Hazard function models were developed for a multiple-dose data set to predict the probability of death through specifying functions of dose response and the time between exposure and the time-to-death (TTD). Among the models developed, the best-fitting survival model (baseline model) is an exponential dose-response model with a Weibull TTD distribution. Alternative models assessed use different underlying dose-response functions and use the assumption that, in a multiple-dose scenario, earlier doses affect the hazard functions of each subsequent dose. In addition, published mechanistic models are analyzed and compared with models developed in this article. None of the alternative models that were assessed provided a statistically significant improvement in fit over the baseline model. The general approach utilizes simple empirical data analysis to develop parsimonious models with limited reliance on mechanistic assumptions. The baseline model predicts TTDs consistent with reported results from three independent high-dose rabbit data sets. More accurate survival models depend upon future development of dose-response data sets specifically designed to assess potential multiple-dose effects on response and time-to-response. The process used in this article to develop the best-fitting survival model for exposure of rabbits to multiple aerosol doses of B. anthracis spores should have broad applicability to other host-pathogen systems and dosing schedules because the empirical modeling approach is based upon pathogen-specific empirically-derived parameters.

Assessment of relative potential for Legionella species or surrogates inhalation exposure from common water uses.

The Legionella species have been identified as important waterborne pathogens in terms of disease morbidity and mortality. Microbial exposure assessment is a tool that can be utilized to assess the potential of Legionella species inhalation exposure from common water uses. The screening-level exposure assessment presented in this paper developed emission factors to model aerosolization, quantitatively assessed inhalation exposures of aerosolized Legionella species or Legionella species surrogates while evaluating two generalized levels of assumed water concentrations, and developed a relative ranking of six common in-home uses of water for potential Legionella species inhalation exposure. Considerable variability in the calculated exposure dose was identified between the six identified exposure pathways, with the doses differing by over five orders of magnitude in each of the evaluated exposure scenarios. The assessment of exposure pathways that have been epidemiologically associated with legionellosis transmission (ultrasonic and cool mist humidifiers) produced higher estimated inhalation exposure doses than pathways where epidemiological evidence of transmission has been less strong (faucet and shower) or absent (toilets and therapy pool). With consideration of the large uncertainties inherent in the exposure assessment process used, a relative ranking of exposure pathways from highest to lowest exposure doses was produced using culture-based measurement data and the assumption of constant water concentration across exposure pathways. In this ranking, the ultrasonic and cool mist humidifier exposure pathways were estimated to produce the highest exposure doses, followed by the shower and faucet exposure pathways, and then the toilet and therapy pool exposure pathways.

Benchmark dose analysis for Bacillus anthracis inhalation exposures in the nonhuman primate.

There is considerable variability in the published lethality values for inhalation exposures of Bacillus anthracis. The lack of consensus on an acceptable dose-response relationship poses a significant challenge in the development of risk-based management approaches for use following a terrorist release of B. anthracis spores. This article reviewed available B. anthracis dose-response modeling and literature for the nonhuman primate, evaluated the use of the U.S. Environmental Protection Agency's Benchmark Dose Software (BMDS) to fit mathematical dose-response models to these data, and reported results of the benchmark dose analysis of suitable data sets. The BMDS was found to be a useful tool to evaluate dose-response relationships in microbial data, including that from B. anthracis exposure. An evaluation of the sources of variability identified in the published lethality data and the corresponding BMDS-derived lethality values found that varying levels of physical characterization of the spore product, differing receptor-specific exposure assumptions, choice of dose metrics, and the selected statistical methods all contributed to differences in lethality estimates. Recognition of these contributors to variability could ultimately facilitate agreement on a B. anthracis dose-response relationship through provision of a common description of necessary study considerations for acceptable dose-response data sets.