Evaluation of two methods for detection of viable Bacillus anthracis simulant spores in maritime environmental samples.

Analytical methods exist to detect biothreat agents in environmental samples during a response to biological contamination incidents. However, the coastal zone facilities and assets of the US Coast Guard (USCG), including response boats in diverse geographical areas and maritime environmental conditions, can pose complex and unique challenges for adapting existing analytical detection methods. The traditional culture (TC) and the rapid viability polymerase chain reaction (RV-PCR) methods were evaluated for their compatibility for maritime environmental surface and grab sample analysis to detect spores of Bacillus thuringiensis subspecies kurstaki (Btk), a surrogate for Bacillus anthracis. The representative samples collected from a USCG installation included surfaces, such as aluminum on boats, nonskid tread on decks of watercraft, computer touchscreens, and concrete piers, and grab samples of boat washdown water, soil, vegetation, and gravel from surrounding areas. Replicate samples were spiked with Btk spores at two to three tenfold increasing levels and analyzed. Out of a total of 150 samples collected and analyzed, the TC method gave 10 false-positive and 19 false-negative results, while the RV-PCR method-based analysis resulted in 0 false-positive and 26 false-negative results. An abundance of microbial background and particulates in some samples interfered with true results, while both methods gave similar results for samples with low microbial background and particulates. Improved and high-throughput sample processing methods are needed for analysis of complex environmental samples.

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.

Comparison of surface sampling methods for an extended duration outdoor biological contamination study.

Bacillus anthracis, the causative agent for anthrax, is a dangerous pathogen to humans and has a history as a bioterrorism agent. While sampling methods have been developed and evaluated for characterizing and clearing contaminated indoor sites, the performance of these sampling methods is unknown for use in outdoor environments. This paper presents surface sampling data for Bacillus atrophaeus spores, a surrogate for B. anthracis, from a 210-day outdoor study that evaluated the detection and recovery of spores using five different sampling methods as follows: sponge sticks, 37-mm vacuum filter cassettes, residential wet vacuums, robotic floor cleaners, and grab samples of soil, leaves, and grass. The spores were applied by spraying a liquid suspension onto the surfaces. Both asphalt and concrete surfaces were sampled by all the surface sampling methods, excluding grab sampling. Stainless steel coupons placed outdoors were additionally sampled using sponge sticks. Sampling methods differed in their ability to collect detectable spores over the duration of the study. The 37-mm vacuums and sponge sticks consistently detected spores on asphalt through day 37 and robots through day 99. The wet vacuums detected spores on asphalt for days 1 and 4, but not again until day 210. On concrete, all samplers detected spores until day 210 except for sponge stick samplers that detected spores only up until the day 99 time point. For all sampling methods, spore recoveries were higher from concrete than from asphalt surfaces. There was no statistically significant difference in recoveries of sponge sticks and 37-mm vacuums from either asphalt or concrete surfaces. Processing of grab samples was challenging due to non-target background microorganisms resulting in high detection limits for the samples.

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.

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.

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.

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.

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.

Toxicity of anthrax toxin is influenced by receptor expression.

Anthrax toxin protective antigen (PA) binds to its cellular receptor, and seven subunits self-associate to form a heptameric ring that mediates the cytoplasmic entry of lethal factor or edema factor. The influence of receptor type on susceptibility to anthrax toxin components was examined using Chinese hamster ovary (CHO) cells expressing the human form of one of two PA receptors: TEM8 or CMG2. Unexpectedly, PA alone, previously believed to only mediate entry of lethal factor or edema factor, was found to be toxic to CHO-TEM8 cells; cells treated with PA alone displayed reduced cell growth and decreased metabolic activity. PA-treated cells swelled and became permeable to membrane-excluded dye, suggesting that PA formed cell surface pores on CHO-TEM8 cells. While CHO-CMG2 cells were not killed by wild-type PA, they were susceptible to the PA variant, F427A. Receptor expression also conferred differences in susceptibility to edema factor.

Neutralizing activity of vaccine-induced antibodies to two Bacillus anthracis toxin components, lethal factor and edema factor.

Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.

Neutralizing antibodies and persistence of immunity following anthrax vaccination.

Anthrax toxin consists of protective antigen (PA) and two toxic components, lethal factor (LF) and edema factor (EF). PA binds to mammalian cellular receptors and delivers the toxic components to the cytoplasm. PA is the primary antigenic component of the current anthrax vaccine. Immunity is due to the generation of antibodies that prevent the PA-mediated internalization of LF and EF. In this study, we characterized sera obtained from vaccinated military personnel. Anthrax vaccine is administered in a series of six injections at 0, 2, and 4 weeks and 6, 12, and 18 months, followed by annual boosters. The vaccination histories of the subjects were highly varied; many subjects had not completed the entire series, and several had not received annual boosters. We developed a simple colorimetric assay using alamarBlue dye to assess the antibody-mediated neutralization of LF-mediated toxicity to the J774A.1 murine macrophage cell line. Recently vaccinated individuals had high antibody levels and neutralizing activity. One individual who had not been boosted for 5 years had low immunoglobulin G antibody levels but a detectable neutralization activity, suggesting that this individual produced low levels of very active antibodies.

Distribution of borreliae among ticks collected from eastern states.

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Borrelia burgdorferi-infected Ixodes species. The disease is typically characterized by an erythema migrans (EM) rash at the site of tick feeding. EM rashes have also been associated with feeding by Amblyomma americanum ticks despite evidence suggesting that they are incompetent vectors for Lyme disease. In 1996, a Borrelia organism only recently cultivated in the laboratory was described in A. americanum ticks and designated B. lonestari. This Borrelia is believed to be the etiologic agent of a novel Lyme-like disease, southern tick associated rash illness (STARI). This study examined ticks collected from eight eastern states to evaluate the epidemiology of B. lonestari, B. burgdorferi, and their tick hosts. Three hundred individual or small pool samples were evaluated from tick genera that included Amblyomma, Ixodes, and Dermacentor. DNA was extracted following tick homogenization and the polymerase chain reaction (PCR) was performed using primers derived from the flagellin gene that amplify sequences from both B. burgdorferi and B. lonestari. Species specific digoxigenin labeled probes were designed and used to differentiate between B. burgdorferi and B. lonestari. Borrelia DNA was detected in approximately 10% of the A. americanum and I. scapularis tick samples, but none was present in any of the Dermacentor samples tested. Positive samples were detected in ticks collected from Kentucky, Maryland, Massachusetts, New Jersey, New York, and Virginia. This is the first known report of B. lonestari from Massachusetts and New York and the first detection in I. scapularis. This suggests that B. lonestari and its putative association with STARI may be more widespread than previously thought.