Sepsis and pathophysiology of anthrax in a nonhuman primate model

Abstract

Studies that define natural responses to bacterial sepsis assumed new relevance after the lethal bioterrorist attacks with Bacillus anthracis (anthrax), a spore-forming, toxigenic gram-positive bacillus. Considerable effort has focused on identifying adjunctive therapeutics and vaccines to prevent future deaths, but translation of promising compounds into the clinical setting necessitates an animal model that recapitulates responses observed in humans. Here we describe a nonhuman primate (Papio c. cynocephalus) model of B. anthracis infection using infusion of toxigenic B. anthracis Sterne 34F2 bacteria (5 x 10(5) to 6.5 x 10(9) CFU/kg). Similar to that seen in human patients, we observed changes in vascular permeability, disseminated intravascular coagulation, and systemic inflammation. The lung was a primary target organ with serosanguinous pleural effusions, intra-alveolar edema, and hemorrhagic lesions. This animal model reveals that a fatal outcome is dominated by the host septic response, thereby providing important insights into approaches for treatment and prevention of anthrax in humans.

Figure 1-6932

Mortality. Animals were infused with B. anthracis Sterne bacteria in doses ranging from 5 × 10⁵ CFU/kg to 6.5 × 10⁹ CFU/kg. All animals except two (*1.6 × 10⁹ and *3.8 × 10⁹ CFU/kg) received antibiotic treatment to more closely mimic a clinical setting.

Figure 2-6932

Toxemia. Plasma levels of anthrax toxin PA were determined by ELISA after infusion of bacteria. Challenge dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3). *P < 0.05; **P < 0.01.

Figure 3-6932

Lung pathology. A: Pleural cavities contained prominent serosanguinous pleural effusions (arrow). B: Hemorrhagic lesions and frothy edematous fluid from the trachea (arrow) is consistent with permeability edema. C: Widespread capillary leakage in lung and intra-alveolar edema, 72-hour survival (H&E). D: Fibrin aggregation (arrow) with generalized epithelial and endothelial damage, 72-hour survival (PTAH). E: Higher magnification view of lung (H&E). F: Higher magnification view of lung (PTAH). G: Severity of lung injury on blinded samples was graded by a pathologist. Tissues were rated according to the severity of the histopathological lesions. The scale ranged from 1 to +4, with 4 being the most severe. Data were grouped according to the dose range. Mean ± SEM. Significant differences from the low-dose challenge (10⁵ to 10⁶ CFU/kg) were determined: *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars = 100 μm. Original magnifications: × 200 (C, D); ×400 (E, F).

Figure 4-6932

Coagulopathy. Hemostatic changes are apparent by loss of fibrinogen (A), prolongation of APTT clotting times (B), elevated D-dimer (C), and reduced platelets (D). Mean ± SEM. Challenge dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3). Significant differences from the low-dose challenge (10⁵ to 10⁶ CFU/kg) were determined: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5-6932

Protein C levels. Citrated plasma was collected at the times indicated, and protein C antigen levels were determined by ELISA as described in the Materials and Methods. Normal (100%) was defined using pooled normal baboon plasma. Challenge dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3). Significant differences from the low-dose challenge (10⁵ to 10⁶ CFU/kg) were determined: *P < 0.05, **P < 0.01.

Figure 6-6932

Vascular permeability. A–C: Increased vascular permeability led to reduced mean systemic arterial pressure (A) accompanied by corresponding increases in respiration (B) and hematocrit (C). Responses were dose-dependent. Mean ± SEM. Challenge dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3). Significant differences from the low-dose challenge (10⁵ to 10⁶ CFU/kg) were determined: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7-6932

Cellular responses. A–C: The changes in WBC counts (A) reflect ongoing neutropenia (B) and lymphocytopenia (C). D: The increase in bands is a typical granulopoietic response to septic challenge. There was no clear dose dependence, but the magnitude of the changes reflected the severity of the challenge. Mean ± SEM. Challenge dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3).

Figure 8-6932

Systemic inflammatory responses. A–D: Increases in TNF-α (A), IL-1β (B), IL-6 (C), and neutrophil elastase (D) occurred after bacterial challenge. Changes were relatively early, transient, and similar to responses observed in other animal models of sepsis. Mean ± SEM. Challenge dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3). Significant differences from the low-dose challenge (10⁵ to 10⁶ CFU/kg) were determined: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9-6932

B. anthracis versus E. coli challenge in the baboon. Plasma levels of IL-8 (A, B), MCP-1 (C, D), MIP-1 (E, F), IL-12p70 (G, H), and IL-12p40 (I, J) were determined by assays from baboons challenged with B. anthracis (top) or E. coli (bottom). Challenge B. anthracis dose in CFU/kg: 10⁵ to 10⁶ (▿, n = 2); 10⁷ (□, n = 3); 10⁸ (•, n = 3); and 10⁹ (▴, n = 3). Challenge E. coli dose in CFU/kg: 10⁸ (▿, n = 3); 10⁹ (□, n = 2); 10¹⁰ (•, n = 3). Mean ± SEM. A–H: There were significant differences between lethal challenges with 10⁸ CFU/kg B. anthracis and 10¹⁰ CFU/kg E. coli; *P < 0.05, ***P < 0.001. I and J: There were significant differences between sublethal challenge with 10⁵ to 10⁶ CFU/kg B. anthracis and 10⁸ CFU/kg E. coli; **P < 0.01.

Figure 10-6932

Immunohistochemistry. Lung tissues from a baboon that received 3.8 × 10⁹ CFU/kg B. anthracis Sterne intravenously (right) or a saline-challenged baboon (control, left) were processed for immunohistochemistry as described in the Materials and Methods. Tissues were stained for TF (green) and TFPI (red) (A, B); inducible nitric oxide synthetase (iNOS, red) and CD68 (green) (C, D); protein tyrosine nitrosylation (red) and CD68 (green) (E, F). G and H: Spleen tissue was stained for apoptosis (green) by TUNEL. Nuclei, blue. a, alveolus. Scale bars = 100 μm.