Pathology and pathophysiology of inhalational anthrax in a guinea pig model

Abstract

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Fig 1

Dose-lethality curve obtained by using the log-transformed estimated inhaled dosage as the predictor variable. The LD₅₀ was determined in three iterations. Groups of animals were exposed to various doses with challenge dose levels in the second and third iterations based on the survival results from the previous iteration(s). In each iteration, guinea pigs were monitored for death twice daily for up to 21 days. The predicted curve from the probit model using data from all three iterations is plotted as a solid line, while the observed lethalities are displayed as symbols by iteration. In addition, the upper and lower 95% confidence bounds on the predicted curve are shown as dashed lines. The two horizontal reference lines correspond to 50 and 90% mortality. The LD₅₀ for all three iterations combined was estimated at 5.01 × 10⁴ spores per animal with a 95% confidence interval of 3.44 × 10⁴ to 7.54 × 10⁴ spores per animal.

Fig 2

Changes in telemetric parameters postchallenge. Nine challenged animals were surgically implanted with telemetry transmitters and monitored for clinical signs of disease. The mean value for each parameter measured by telemetry was computed every 15 min for 24 h (00:00, 00:15,…, 23:45) prechallenge (baseline). The data collected postchallenge were then baseline adjusted according to the associated clock time. In these graphs, the mean differences that were significantly greater than zero are indicated by black squares, while the mean differences that were significantly less than zero are indicated by black triangles. The parameters monitored included temperature (A), heart rate (B), MAP (C), pulse pressure (D), diastolic BP (E), and systolic BP (F).

Fig 3

Bacterial and toxin levels postchallenge. (A) Quantitative bacteremia, A 100-μl volume of whole blood was plated in triplicate on TSA. In addition, 10-fold serial dilutions were performed by transferring 100 μl of whole blood or the previous dilution into 900 μl of PBS. A 100-μl volume of each dilution prepared was plated in triplicate on TSA. Plates were incubated at 37°C for 16 to 24 h, bacterial colonies were enumerated, and the corresponding concentration (CFU/ml) was calculated. (B) PA levels measured by ELISA. Plates were read, and the data were analyzed by using a 4PL model to fit the eight-point calibration curve. The PA concentrations in samples were determined by computer interpolation from the plot of the reference standard curve data.

Fig 4

Histopathological findings. Tissue samples were preserved in 10% neutral buffered formalin, paraffin embedded, processed to slides, and stained with HE. Representative histopathological findings are shown. (A) Lymph node sinuses and extravascular tissues are filled with blood (hemorrhage), and only remnants of lymphoid follicles remain (arrows). HE staining, ×4 magnification. (B) Brain, meninges. The blood vessel shown contains large, rod-shaped bacteria consistent with anthrax bacilli (arrows). HE staining, ×10 magnification. (C) Alveoli filled with fibrin and edema (arrows) including pulmonary intravascular B. anthracis (arrowheads). HE staining, ×40 magnification. (D) Renal vessels containing anthrax bacilli (arrows). HE staining, ×40 magnification. (E) Spleen tissue showing sinusoidal anthrax bacilli and degenerate and viable heterophils (arrows). HE staining, ×40 magnification. (F) Heart tissue showing anthrax bacilli within coronary vessels (arrows). HE staining, ×10 magnification.