Clinical and Pathological Findings Associated with Aerosol Exposure of Macaques to Ricin Toxin

Abstract

Ricin is a potential bioweapon that could be used against civilian and military personnel. Aerosol exposure is the most likely route of contact to ricin toxin that will result in the most severe toxicity. Early recognition of ricin exposure is essential if specific antidotes are to be applied. Initial diagnosis will most likely be syndromic, i.e., fitting clinical and laboratory signs into a pattern which then will guide the choice of more specific diagnostic assays and therapeutic interventions. We have studied the pathology of ricin toxin in rhesus macaques exposed to lethal and sublethal ricin aerosols. Animals exposed to lethal ricin aerosols were followed clinically using telemetry, by clinical laboratory analyses and by post-mortem examination. Animals exposed to lethal aerosolized ricin developed fever associated with thermal instability, tachycardia, and dyspnea. In the peripheral blood a marked neutrophilia (without immature bands) developed at 24 h. This was accompanied by an increase in monocytes, but depletion of lymphocytes. Red cell indices indicated hemoconcentration, as did serum chemistries, with modest increases in sodium and blood urea nitrogen (BUN). Serum albumin was strikingly decreased. These observations are consistent with the pathological observations of fluid shifts to the lungs, in the form of hemorrhages, inflammatory exudates, and tissue edema. In macaques exposed to sublethal aerosols of ricin, late pathologic consequences included chronic pulmonary fibrosis, likely mediated by M2 macrophages. Early administration of supportive therapy, specific antidotes after exposure or vaccines prior to exposure have the potential to favorably alter this outcome.

Keywords: aerosol exposure; biodefense; ricin; syndromic diagnosis; telemetric monitoring; toxin.

Figure 1

Telemetric monitoring of vital signs following aerosol exposure to ricin toxin. Changes in the mean hourly values for the parameters are plotted against time for each animal. Alterations in body temperature in non-exposed macaques, showing normal diurnal variation (A), and following ricin exposure (B); heart rate (C) and respiratory rate (D) are shown. The fever (B) and heart rate (C) curves, shown here as individual animals, were averaged and shown as the controls in Figure 2 of reference [21].

Figure 2

Hematologic changes in individual macaques. White cell counts (A–D) and red cell indices (E–G) are compared prior to and 20–22 h following ricin exposure.

Figure 3

Changes in selected clinical chemistry values in individual macaques. Serum protein concentrations are shown at (A–C), parameters demonstrating possible hemoconcentration on the (D–F).

Figure 4

Histological examination of lung tissue one day following exposure to lethal (acute) and 20 days following sublethal (chronic) concentrations of ricin toxin. Acute changes were characterized by massive alveolar edema, infiltrations of mixed inflammatory cells and marked alveolar necrosis (A,C,E). Chronic changes were characterized by areas of interstitial fibrosis which frequently obliterate functional alveolar spaces (B,D,F). Scale bars in the lower right corner of each figure represent 300 µm (A,B) or 50 µm (C–F).

Figure 5

Confocal microscopy to identify subset of macrophages found within the intra-alveolar space 20 days following sublethal exposure to aerosolized ricin. Co-expression of CD206, the mannose receptor (red with CD68, CD163, or MAC387 were visualized to see the changes in the distribution of specific subsets of macrophages in relation to their location in lung. Almost all intra-alveolar macrophages co-expressed CD206 with CD68 and CD163 (orange signal in A and B). MAC387 (green signal panel C) was present exclusively in the interstitial/intravascular macrophages and was absent in which CD 206+ intra-alveolar macrophages. Cell nuclei were stained blue with DAPI.