Fig 1.
Nose-only rabbit aerosol exposure test system.
The diagrammed laboratory-scale system was housed in a class III biological safety cabinet (BSC III) for safe operations. Animal exposure to aerosolized spores was performed as previously described [25]. A 3-jet Collison nebulizer was loaded with Bcbva spores diluted to an approximate dosage target. The test system was operated under passive negative pressure conditions in which air was continuously drawn through the system by the APS and vacuum from each mask (1, 2) during animal exposure. The aerosolized particles generated from the Collison were counted and sized by the APS and aerosolized spores quantified from filters after sampling the exposure air stream at one liter per minute. Image created in Microsoft PowerPoint using drawing tools in PowerPoint.
Fig 2.
Dose-response in rabbits exposed by inhalation to Bcbva CA and CI spores.
Particle size distributions of purified spores during aerosol exposures were measured by the APS. The mass median aerodynamic diameter (MMAD) for each set of animal exposures is indicated by individual dots (A). The horizontal line represents the replicate mean and corresponding geometric standard deviation. Probit dose-response (solid line) with 85% confidence intervals (dashed lines) are shown for Bcbva CA (B) and CI (C).
Table 1.
Dose-dependent median time to death following inhalation exposure to Bcbva CA and CI. Log-Rank Mantel Cox Test was performed to identify differences in time to death among the administered doses. Data were derived from 8 animals per dose of Bcbva CA and CI and 10 animals for Ames A2084. Since multiple deaths/dose group are required to determine a median time to death, only the data for those doses are listed. The B. anthracis data were derived from a previous study [25]. Since dose does not influence time to death for B. anthracis challenge, the dose range and mean time to death are reported.
Fig 3.
Representative temperature responses in rabbits exposed by inhalation to Bcbva CI spores.
Significant increase in body temperature (SIBT) and Time of Death (ToD) are indicated with arrows and the value is provided. SIBT is defined as 3x the standard deviation of the baseline temperature. The orange and blue dotted lines indicate 3x the standard deviation above and below the baseline temperature, respectively. Blue temperature readings are the baseline measurements prior to exposure, and red temperature measurements are following exposure. SD indicates standard deviation. A. Survivor, dose 2.79 X 105 CFU. B. Non-survivor with a short duration between SIBT and death, dose 3.63 X 106 CFU. C. Non-survivor with a long duration between SIBT and death, dose 4.31 X 105 CFU.
Fig 4.
SIBT and time to death in rabbits exposed by inhalation to Bcbva CA and CI spores.
The time to SIBT following aerosol exposure was recorded for each animal and plotted as the percentage of animals with normal body temperature per dosage group (A, B). The time of death for animals that succumbed to infection was plotted as percent survival per dosage group (C, D). Two groups of 8 animals were challenged with 1.8 X 105 CFU Bcbva CI. These groups were combined for data analysis (16 animals in this group).
Table 2.
Terminal bacterial burden in animals infected with Bcbva. Animals that succumbed to infection in the LD50 studies (n = 23 for CA; n = 25 for CI) were sampled, and mean CFU/ml or g determined for blood, BAL, liver, lung, and spleen tissues. Six survivors were sampled as controls and bacteria were not observed in any of the assayed tissues or fluids at 28 days post-exposure. Data shown are mean with lower and upper 95% confidence intervals in brackets.
Fig 5.
Longitudinal evaluation of bacteremia following Bcbva inhalation spore challenge.
Symbols indicate each data point, and time points are relative to the time of death in days (d). Mean titers (horizontal line) are shown. The sampling results are shown for animals with at least two consecutive days of sampling (n = 16 for CA; n = 21 for CI). Only quantifiable titers are shown. The absence of a data point indicates no detectable bacterial growth observed from the sample of corresponding animals.
Fig 6.
Longitudinal evaluation of Bcbva CA virulence factor expression.
Hyaluronic acid (A) and protective antigen (B) concentrations were measured in serum samples each day for five days following exposure to Bcbva CA spores. Symbols indicate individual data points. Horizontal line indicates the mean. Time points are relative to the time of death (d) for animals that succumbed to aerosol exposure within the first five days of the observation period. The dashed horizontal lines indicate the mean virulence factor expression levels in all samples from survivors for the first 5 days following aerosol exposure, since no statistical increase was observed for survivors during this period. For non-survivors, statistical significance of the level of virulence factor expression was determined using ANOVA analysis followed by a Tukey’s multiple comparisons test as described in the methods. * indicates a P-value of< 0.05.
Fig 7.
Longitudinal evaluation of Bcbva CI virulence factor expression.
Hyaluronic acid (A) and protective antigen (B) concentrations were measured in serum samples each day for five days following exposure to Bcbva CI spores. Symbol indicates data point. Horizontal line indicates the mean. Time points are relative to the time of death (d) for the analyzed animals that succumbed to aerosol exposure within the first five days of the observation period. The dashed horizontal lines indicate the mean virulence factor expression levels in all samples from survivors for the first 5 days following exposure since no statistical increase was observed for survivors during this period. For non-survivors, statistical significance of the level of virulence factor expression was determined using ANOVA analysis followed by a Tukey’s multiple comparisons test as described in the methods. * indicates a P-value of < 0.05.
Fig 8.
In vitro versus terminal in vivo virulence factor expression.
Data represents the mean and standard deviation of the analyzed biological replicates (three replicates for in vitro; 10 Bcbva CA and 14 Bcbva CI replicates each for in vivo) for hyaluronic acid (A), protective antigen (B), and PDGA capsule (C) at stationary growth (in vitro) or in terminal blood samples (in vivo), which would constitute maximal growth in the respective conditions. The amount of HA in the B. anthracis samples was not statistically different from a negative control. HA was not detected in serum collected from animals prior to challenge with Bcbva CA or CI.
Table 3.
Summary of MCM efficacy and outcomes following Bcbva CA or CI aerosol exposure. MCM were administered as described in Materials and Methods. *Indicates animal succumbed immediately after treatment. Animal data was censored. #Indicates a telemetry transponder failed, and no temperature data was collected. βIndicates SIBT occurred following completion of treatment course. ^Indicates quantified values were not statistically different than pre-administration samples via pair-wise comparison. NA indicates not applicable. †Indicates seroconversion occurred before exposure.
Fig 9.
Temperature responses and time to death as a function of treatment following Bcbva CA aerosol exposure.
Time to SIBT (A), time to death (B), magnitude of SIBT (peak and mean; C and D, respectively), number of SIBT (E), and cumulative duration of SIBT (F)] are indicated. Post indicates post-exposure prophylaxis (PEP). The time to SIBT for post levofloxacin-treated animals was determined from cessation of treatment. If the animal did not display SIBT or survived the 28-day observation period, 672.00 h was used as the time to SIBT and death, respectively. + Indicates treatment occurred. – Indicates treatment did not occur. One-way ANOVA was performed followed by a Dunnett’s Test to perform pair-wise comparisons between the MCM treatment groups and control. * Indicates P < 0.05. ** Indicates P < 0.01. *** Indicates P < 0.001. **** Indicates P < 0.0001. Only statistically significant comparisons are shown.
Fig 10.
Temperature responses and time to death as a function of treatment following Bcbva CI aerosol exposure.
Temperature response [time to SIBT (A), time to death (B), magnitude of SIBT (peak and mean; C and D, respectively), number of SIBT (E), and cumulative duration of SIBT (F)] are indicated. Post indicates post-exposure prophylaxis (PEP). The time to SIBT for levofloxacin treated animals was determined from cessation of treatment. If the animal did not display SIBT or survived the 28-day observation period, 672.00 used as the time to SIBT and death, respectively. + Indicates treatment occurred. – indicates treatment did not occur. One-way ANOVA was performed followed by a Dunnett’s Test to perform pair-wise comparisons between the MCM treatment groups and control. * Indicates P < 0.05. ** Indicates P < 0.01. *** Indicates P < 0.001. **** Indicates P < 0.0001. Only statistically significant comparisons are shown.
Fig 11.
Terminal bacterial burden in animals that succumbed following the cessation of levofloxacin treatment and non-treated controls.
The CFU/mL in blood or broncho-alveolar lavage (BAL) samples and CFU/g of lung, liver, or spleen tissues of terminal untreated animals and animals that died after completion of levofloxacin treatment were quantified for Bcbva CA (A), or Bcbva CI (B). + Indicates treatment. – Indicates no treatment. Pair-wise comparisons were performed with a 2-way Student’s T-test comparing bacterial titers between tissues harvested from levofloxacin-treated animals and control. * Indicates P < 0.05. ** Indicates P < 0.01. Only statistically significant comparisons are shown.
Fig 12.
Quantification of virulence factors in MCM-treated animals following aerosol exposure to Bcbva CA and CI.
Hyaluronic acid (A) and protective antigen (B) were quantified in serum samples upon death or from survivors at the end of the observation period. Symbols indicate individual data points. Horizontal line indicates the mean. For control and levofloxacin-only treated animals the data values are from non-survivor terminal samples. For dual treatment with PEP and Post-AVA, and pre-AVA animals, the data values represent quantification of day 28 samples since no animals succumbed during the observation period. Collectively, comparisons were made between survivors and non-survivors as a function of treatment regimen. + Indicates treatment. – Indicates no treatment. One-way ANOVA was performed followed by a Tukey’s Test to perform pair-wise comparisons between the MCM treatment groups and control. * Indicates P < 0.05. ** Indicates P < 0.01. *** Indicates P < 0.001. **** Indicates P < 0.0001. Only statistically significant comparisons are shown.
Fig 13.
Seroconversion against protective antigen.
Antibodies that bind to protective antigen were measured in serum samples from rabbits exposed to Bcbva and treated with medical countermeasures (A). Seroconversion in MCM-treated animals. Only the animals that seroconverted are shown. Results for Bcbva CA and CI experiments were combined since a statistical difference was not observed as a function of challenge strain by ROUT outlier analysis (post-levofloxacin survivors) and pair-wise comparisons with a two-way Student’s T-test (post-levofloxacin and AVA; Pre-AVA). The survival outcome for each treatment group is indicated. + Indicates treatment occurred. – Indicates treatment did not occur. Symbols indicate individual data points. Horizontal line indicates the mean. One-way ANOVA was performed followed by a Tukey’s Test to perform pair-wise comparisons between the MCM treatment groups, except for the post-levofloxacin non-survivor group (too few data point for statistical analysis). Only statistically significant comparisons are shown. Blood serum samples from INHLD50 experiments were quantified for antibody that binds protective antigen (B). Pair-wise comparisons were performed with a 2-way Student’s T-test comparing survivors and non-survivors. NS indicates non-survivors. S indicates survivors.