Evaluation of Plasmodium berghei for Endotoxin by the Limulus Lysate Assay

Abstract

The presence of endotoxin in the malaria parasite has long been suspected but has not been directly demonstrated by the Limulus amoebocyte lysate (LAL) assay or other assays. Tests for endotoxins have been done on plasma of infected individuals but not on the parasites themselves (Glew and Levin, 1975, Proc. Soc. Exp. Bio. Med. 148: 508-510). Recently, Tubbs (1980, Trans. R. Soc. Trop. Med. Hyg. 74: 121-123) detected endotoxin activity in the plasma of mice infected with Plasmodium berghei and in patients infected with P. falciparum using the LAL assay, and suggested that the endotoxin was derived from either the parasites or the endogenous bacteria in the gut. By assaying intact plasmodia washed free of contaminating host cells and plasma, it is possible to determine whether the plasmodia are the source of the endotoxin. We obtained free P. berghei using ultrasonic energy and tested the plasmodia for endotoxin activity using the LAL assay. The results obtained are the subject of this report. Groups of mice and rats were infected by the intraperitoneal injection of approximately 106 P. berghei parasites, and later the blood was collected aseptically by cardiac puncture when parasitemia reached 40%. The blood was transferred to pyrogen-free, sterile, heparinized tubes and washed three times in sterile, pyrogen-free 0.9% NaCl. Blood from uninfected mice was treated in an identical manner and served as a control. The blood specimens were then sonicated as previously described (Prior and Kreier, 1972, Exp. Parasitol. 32: 239-243). The continuous-flow chamber and sonicator probe tip were rendered pyrogen-free by heating at 160 C overnight. All equipment and solutions were tested for endotoxin contamination with the LAL test before use. Following sonication the specimens were centrifuged to harvest the plasmodia, and then adjusted to 25% packedcell volume. The specimens were lysed by three cycles of freeze-thawing and assayed quantitatively for the presence of endotoxin using the microdilution LAL assay (Prior and Spagna, 1979, J. Clin. Microbiol. 10: 394395). The minimum sensitivity of the Limulus lysate utilized in the microdilution assay (courtesy of Mallinckrodt Inc., St. Louis, Missouri) was 0.06 ng/ml of LPS using the E. coli reference standard (lot EC-2, Bureau of Biologics, Food and Drug Administration). Aliquots of free parasite and control samples were seeded with known quantities of the E. coli endotoxin and assayed. Because certain heat-labile protein substances may give positive LAL test results, portions of the samples were heated at 100 C for 5 min and then tested for endotoxin activity. Parasites and control samples were also cultured on blood agar aerobically and anaerobically to detect any possible bacterial contamination. The results obtained in four experiments using the LAL assay on the lysed parasite suspensions and controls are shown in Table I. A reaction suggestive of endotoxin was ob-