Kinetics of lethal factor and poly-D-glutamic acid antigenemia during inhalation anthrax in rhesus macaques

Abstract

Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic gamma-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean +/- standard error [SE] between animals) were low at 24 h postchallenge (0.03 +/- 1.82 ng/ml), increased at 48 h to 39.53 +/- 0.12 ng/ml (phase 1), declined at 72 h to 13.31 +/- 0.24 ng/ml (phase 2), and increased at 96 h (82.78 +/- 2.01 ng/ml) and 120 h (185.12 +/- 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 +/- 2 ng/ml), declined at 72 h (14 +/- 0.2 ng/ml), and then increased at 96 h (3,401 +/- 8 ng/ml) and 120 h (6,004 +/- 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% +/- 0.13%) to 48 h (75.6% +/- 0.08%) and declined at 72 h (62.4% +/- 0.05%). The 72-h declines may establish a "go/no go" turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.

FIG. 1.

Kinetics profile for LF and PGA in rhesus macaques. (A) The geometric mean and individual values of LF (ng/ml) for each animal infected with B. anthracis Ames strain were plotted on a log₁₀ scale for each time point in hours to compare the kinetic profiles over time. (B) The geometric mean and individual values of PGA (ng/ml) were plotted on a log₁₀ scale for each time point. The same symbols are used for panels A and B: animal A, ⋄; animal B, □; animal C, ○; animal D, ▵; animal E, ×; and geometric mean, •. The negative value for PGA at 24 h was assigned a value of the limit of detection divided by two of 1.125 ng/ml according to Hornung et al. (13a).

FIG. 2.

Correlation between levels of LF and PGA in sera from rhesus macaques infected with B. anthracis Ames strain. LF and PGA results collected after 24 h were paired, and the 51 paired values were transformed using log₁₀ and analyzed using least squares regression analysis, and the correlation coefficient was calculated. The slope was significantly different from 0 (P < 0.0001), and the correlation coefficient (r²) was 0.869.

FIG. 3.

Kinetic profile of immune responses in rhesus macaques infected with B. anthracis Ames strain. Percent neutrophils (A), percent lymphocytes (B), and the N/L ratio (C) were plotted over time for the geometric mean and individual values for all five animals. The same symbols are used for panels A, B, and C: animal A, ⋄; animal B, □; animal C, ○; animal D, ▵; animal E, ×; and geometric mean, •. The time points for −42 days (−42D) and 2 h are not to scale.