Richard B. Prior

Plasmodium berghei freed from host erythrocytes by a continuous-flow ultrasonic system

Abstract Ultrasound was used to free Plasmodium berghei from host erythrocytes. Debris formation, parasite damage, and red cell-membrane entrapment of parasites were minimized by using a continuous-flow system of sonication. The free parasites were harvested by differential centrifugation, and low-power thin-section electron microscopy showed the effectiveness of the procedure. The free parasites were used as antigens in complement-fixation tests, and they reacted well with sera from rats recovering from Plasmodium berghei infection.

Evaluation of Plasmodium berghei for endotoxin by the Limulus lysate assay.

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-

Application of the limulus lysate assay in evaluation of disseminated gonorrhea in women.

The limulus lysate assay was utilized as a diagnostic adjunct in the evaluation of three cases of disseminated gonorrhea in women. Although not a specific test for Neisseria gonorrhoeae, the limulus lysate assay, when used with properly diluted endocervical samples, gave results that correlated with conventional diagnostic techniques. If the advantages and limitations of the limulus lysate assay become fully appreciated, it may serve as a useful clinical tool for diagnosis of this syndrome.

Detection of polysaccharide cell wall antigen of Neisseria gonorrhoeae in a rabbit model by counterimmunoelectrophoresis.

A rabbit chamber model was developed and inoculated with 10(9) colony-forming units (cfu) of viable Neisseria gonorrhoeae to determine whether the lipopolysaccharide-derived Gc2 polysaccharide cell wall antigens could be detected by counterimmunoelectrophoresis (CIE). Four hours after inoculation, a polymorphonuclear leukocyte response was noted in the chambers; this response was followed by progressive phagocytosis of the organisms and a fall in number of cfu/ml. All visible bacteria were intracellular, and chamber fluids were sterile 6 hr after inoculation. Use of sero specific antisera permitted detection by CIE of the Gc2 polysaccharide antigen in sera of all rabbits within 48 hr after inoculation of the chambers, whereas blood cultures remained sterile throughout the experiment. At 2-6 hr after inoculation, the Gc2 polysaccharide antigen was also detected as a single precipitin band in the chamber fluid of inoculated rabbits. At 24 hr the precipitin band was not observed; rather, a halo above the antigen well was noted. The halo was found to be a nonspecific complex containing the Gc2 polysaccharide antigen and no antibody. In the rabbit model studied, CIE was sufficiently sensitive to detect concentrations of the Gc2 polysaccharide antigen of greater than or equal to 0.97 microgram/ml in serum and chamber fluid.

Proposed Mezlocillin Disk Control Values for Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853

Individual test, accuracy, and precision control values were determined for the 75- µ g mezlocillin disk with Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.