Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immuno-PCR

Abstract

Background: Ricin (also called RCA-II or RCA(60)), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis. Several in vitro methods have been designed for ricin detection in complex food matrices in the event of intentional contamination. Recently, a novel Immuno-PCR (IPCR) assay was developed with a limit of detection of 10 fg/ml in a buffer matrix and about 10-1000-fold greater sensitivity than other methods in various food matrices.

Methods and findings: In order to devise a better diagnostic test for ricin, the IPCR assay was adapted for the detection of ricin in biological samples collected from mice after intoxication. The limit of detection in both mouse sera and feces was as low as 1 pg/ml. Using the mouse intravenous (iv) model for ricin intoxication, a biphasic half-life of ricin, with a rapid t(1/2)α of 4 min and a slower t(1/2)β of 86 min were observed. The molecular biodistribution time for ricin following oral ingestion was estimated using an antibody neutralization assay. Ricin was detected in the blood stream starting at approximately 6-7 h post- oral intoxication. Whole animal histopathological analysis was performed on mice treated orally or systemically with ricin. Severe lesions were observed in the pancreas, spleen and intestinal mesenteric lymph nodes, but no severe pathology in other major organs was observed.

Conclusions: The determination of in vivo toxicokinetics and pathological effects of ricin following systemic and oral intoxication provide a better understanding of the etiology of intoxication and will help in the future design of more effective diagnostic and therapeutic methods.

Figure 1. Sections of lymph nodes in mice ip treated with pure ricin were stained with hematoxylin and eosin.

A. Lymph node at day one post-intoxication. Several tingible body macrophages (arrows) are present within a follicle consistent with lymphoid reactivity. B. Lymph node at day two post-intoxication. There is moderate lymphoid necrosis characterized by clusters of small, dark basophilic, round apoptotic bodies (arrows) admixed with intact lymphocytes. C. Lymph node from non-treated control mouse for comparison. Bar = 100 µm.

Figure 2. Standard curves for IPCR detection of ricin in sera and feces.

Ct values were plotted against log ricin concentrations (pg/mL) for three replicate samples in mouse sera (A) and feces (B). The linear regression of the calibration curve has a correlation coefficient of r² = 0.99. The dashed lines are the upper and lower 95% confidence limits. The solid horizontal thick line denotes the average blank Ct value, with the standard deviation drawn at each end of this line. The solid horizontal thin line intersecting the linear regression line indicates the detection threshold of the IPCR assay as defined in the text.

Figure 3. Serum toxicokinetics of ricin.

A. Concentrations of ricin in mouse sera over 480 min post -intoxication. Ricin content was determined by IPCR. B. Concentrations of ricin in mouse sera from 2 to 30 min post-intoxication illustrating early and rapid sequestration of ricin with a t_1/2α half-life of 4 min. Each point in the graph represents the mean value ± SEM. N≥5.

Figure 4. Neutralization of ricin with polyclonal ricin antibodies.

Survival percentage of mice dosed iv (A) or ig (B) with ricin were plotted over 160 h post-intoxication. Goat anti-ricin antibodies were introduced iv either prior-to or post-intoxication at indicated times. PBS was injected into mice as no treatment control. Comparison of survival curves with log rank test yielded statistical significance of p<0.001 and p<0.0144 for A and B respectively.