Figure 1.
Viability of B. pseudomallei (Bp) and B. mallei (Bm) upon use of the MicroSprayer® and delivery of viable organisms into the murine lungs.
Panel A: Bacteria were suspended in PBS to an optical density of 1X106 bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL (black bars). The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into 1 mL of sterile PBS, which was serially diluted and plated onto agar medium to determine the number of viable bacteria (grey bars). Results are expressed as the mean (± standard error) colony forming units (CFU). These experiments were performed in triplicate on 2 separate occasions. Panel B: Bacteria were suspended in PBS to optical densities of 1X106 (Dose 1), 1X105 (Dose 2), and 1X104 (Dose 3) bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL (black bars). The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into the lungs of mice (n=3 per dose). Thirty minutes post-inoculation, the mice were euthanized and their lungs were collected, homogenized, diluted, and plated onto agar medium to determine bacterial loads (grey bars). Results are expressed as the mean (± standard error) CFU. These experiments were performed on at least 2 separate occasions. The Mann-Whitney test was used to compare the number of viable organisms in 50 µL of bacterial suspension (i.e. before using the MicroSprayer®; black bars) to that in 1 mL PBS (panel A) or lung homogenates (panel B) after the use of the MicroSprayer® (i.e. grey bars). No statistically significant differences were noted.
Figure 2.
LD50 of B. pseudomallei 1026b (Bp) and B. mallei ATCC23344 (Bm) after inoculation with the MicroSprayer®.
The MicroSprayer® was used to deliver the indicated number of bacterial CFU into the lungs of BALB/c mice. Animals were then monitored for clinical signs of illness and morbidity. Survival data were analyzed with the Kaplan-Meier method and the LD50 values were calculated according to Reed and Muench [55]. The number of animals/group is shown in parentheses. Control mice were inoculated with 50 µL of PBS using the MicroSprayer®. Panels A and B show 2 separate experiments to determine the LD50 of B. pseudomallei strain 1026b. Panels C and D show 2 independent experiments to determine the LD50 of B. mallei ATCC23344. With the exception of the survival curves for PBS and the lowest inoculating CFU dose, survival curves were found to be statistically different using the Logrank test for trend (panels A through D, p ≤ 0.05).
Figure 3.
Clinical progression of disease after inoculation with the MicroSprayer®.
Figure 4.
Bacterial loads in lungs, spleen and blood after inoculation with the MicroSprayer®.
Bacteria were suspended in PBS to optical densities of 1X106 (Dose 1), 1X105 (Dose 2), and 1X104 (Dose 3) bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL. The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into the lungs of mice (n=15 per dose). At the indicated time points post-inoculation, mice (n=3 per dose) were euthanized and tissues were collected, homogenized, diluted, and plated onto agar medium to determine bacterial loads. Results are expressed as the mean (± standard error) total CFU/per organ (panels A, B, C, D) and mean (± standard error) CFU/ml of blood (panels E and F). These experiments were performed on at least 2 separate occasions. Bp= B. pseudomallei 1026b, Bm=B. mallei ATCC23344. Both organisms were first detected in the spleen at 24-hr post-challenge (panels C and D). Bp disseminated to the organ in significantly greater numbers than Bm at the challenge doses of 10 and 1 LD50 (Wilcoxon Signed Rank test p<0.0001).
Figure 5.
Bacterial loads in bronchoalveolar lavage fluids (BALF) and lungs after inoculation with the MicroSprayer®.
Bacteria were suspended in PBS to optical densities of 1X106 (Dose 1), 1X105 (Dose 2), and 1X104 (Dose 3) bacteria/mL, serially diluted, and plated onto agar medium to calculate the number of viable organisms in 50 µL. The MicroSprayer® was then used to deliver 50 µL of bacterial suspensions into the lungs of mice (n=3 per dose). Control mice were inoculated with 50 µL of sterile PBS. Seventy-two hours post-inoculation, the animals were euthanized and tissues (BALF, lungs) were collected, homogenized, diluted, and plated onto agar medium to determine bacterial loads. Results are expressed as the mean (± standard error) total CFU/per tissue. Bp= B. pseudomallei 1026b, Bm=B. mallei ATCC23344. Black bars= lung homogenates, grey bars=BALF.
Figure 6.
Cytologic evaluation of BALF from infected mice.
Bronchoalveolar lavage fluids from control and infected animals (see Figure 5) were concentrated onto a glass slide, fixed with methanol, air-dried, stained with modified Wright-Giemsa, and examined by microscopy (100X objective). Panel A shows a representative field of a sample collected from a mouse inoculated with sterile PBS (control). These control samples are low in cellularity and consist of occasional macrophages (arrow) and ciliated, columnar respiratory epithelial cells (inset). Panel B shows a representative field of a sample from a mouse infected with 104 B. mallei bacteria. Large numbers of degenerate neutrophils (block arrows) and foamy macrophages (arrows) are seen admixed with cellular debris and occasional red blood cells. Medium-sized bacilli are seen phagocytosed by neutrophils (asterisk). Extracellular bacteria are also present (arrowheads). Panel C is a representative field of a sample from mouse infected with 103 B. mallei cells, and Panel D is a representative field of a sample from mouse infected with 103 B. pseudomallei CFU. In both panels, moderate numbers of degenerate neutrophils and foamy macrophages are present, along with intracellular bacilli, illustrating the similar type and magnitude of inflammation elicited by the two species at the same dose. The inset in Panel C demonstrates that neutrophils contain bacilli adhered to their surface and within the lumens of intracytoplasmic vacuoles.
Figure 7.
ELISA with sera from mice that survived aerosol challenge with lethal doses of B. pseudomallei 1026b (Bp) and B. mallei ATCC23344 (Bm).
Serum samples were serially diluted and placed in duplicate wells of plates coated with CPS (panel A), OPS (panel B), His-tagged BoaA (panel C), His-tagged BPSL1631-BMA1027 (panel D), and His-tagged BPSS0908-BMAA1324 (panel E). Goat α-mouse Abs conjugated to HRP were used as secondary Abs. The y-axis shows absorbance at a wavelength of 650 nm, which is indicative of antibody binding to antigens coating the plates. The x-axis represents serial two-fold dilutions of sera starting at 1:100 to 1:12,800. The results are expressed the mean absorbance (± standard error). Open diamonds show sera from mice that survived challenge with Bm. Closed circles show sera from mice that survived challenge with Bp. Blue squares represent sera from control mice that were inoculated with 50 µL of PBS using the MicroSprayer®.