Schizont-infected cell enrichment in rodent malaria.

Mouse erythrocytes parasitized with Plasmodium berghei or Plasmodium yoelii were separated by ultracentrifugation using preformed isodensity gradients of the discontinuous type. Three fractions were obtained following centrifugation, the upper of which contained greater than 90% of all schizont-infected cells added to the gradient. The gradient material, Stractan II, is an arabinogalactan polysaccharide and appears to yield results similar to those available using gradients of bovine serum albumin. Cells are not altered morphologically or physiologically (as assessed by infectivity) by the treatment. The asexual stages of malarial parasites are often described as having an obligate intracellular habitat. Such descriptions are fundamentally inaccurate, due to the cyclical nature of the parasites' asexual development, which includes periodic schizont rupture and merozoite release into the host plasma. The merozoite is thus an extracellular stage, and appears to be highly specialized for extracellular survival, albeit for a brief interval (Aikawa, 1971). The rodent parasites Plasmodium berghei and P. yoelii are attractive models for the study of mammalian malaria; virulent strains produce overwhelming infections in many strains of mice. However, at no time do these parasites exhibit a degree of synchrony comparable to that shown by the simian parasite P. knowlesi, and they are therefore limited in their potential to provide unequivocal information about the biology of the merozoite. Numerous investigators have attempted to purify the schizont-infected cells (hereafter referred to as "schizonts") by exploiting a property initially discovered by Bass and Johns (1915-16). These workers significantly concentrated P. vivax-infected cells by centrifuging the cells and collecting the parasite-rich upper elements of the packed cell column. These findings were refined by Ferrebee and Geiman (1946), who concluded that there is an inverse relationship between cell density and age of the parasite (P. vivax). These workers used density gradient centrifugation in bovine serum albumin (BSA), and this method remains to date the most satisfactory for the centrifugal enrichment of parasitized cells. The method Received for publication 17 January 1976. Revised 5 March 1976. 664 works well with P. berghei also; Rowley et al. (1967) used continuous linear gradients of BSA and obtained excellent separation of the various stages of parasitized cells. Unfortunately, BSA is prohibitively expensive for most investigators, and thus alternative materials have been investigated for use as high-density media for parasite concentration. For various reasons (see Discussion) none of the alternative materials in use are judged to be suitable in comparison to BSA. Corash et al. (1974) have recently reported a new gradient material for use in the centrifugal fractionation of human erythrocytes. The material, Stractan II, is an arabinogalactan polysaccharide of 30,000 daltons. Corash et al. (loc. cit.) reported solutions of this material to have comparable properties to BSA when used as a gradient material, and to be inexpensive in comparison to BSA. This study reports the details of the use of Stractan gradients for the differential fractionation of parasitized cells of P. berghei and P. yoelii. The method produces significant fractionation of the various erythrocytic stages, and yields cell suspensions which are intact, both morphologically and physiologically, as measured by their ability to establish patent i fections in susceptible mice. MATERIALS AND METHODS Plasmodium berghei (Strain NYU-2, kindly supplied by Dr. Carter L. Diggs, Walter Reed Army Institute of Research, Washington, D. C.) was maintained in random-bred adult male Swiss mice (Texas Inbred Mice Co., Houston, Tex.). The virulent line of strain 17X of P. yoelii (kindly supplied by the late Dr. M. Yoeli, New York University) was maintained in similar fashion. Both parasite strains are routinely lethal in this strain This content downloaded from 207.46.13.149 on Mon, 03 Oct 2016 05:44:46 UTC All use subject to http://about.jstor.org/terms McALISTER AND GORDON-SCHIZONT-INFECTED CELL ENRICHMENT IN RODENT MALARIA 665 of mice; an infective dose of 5 X 105 parasitized cells given i.p. gives lethal parasitemias by day 7 p.i.; each log increase in infective dose gives a decreased mean survival time of approximately 24 hr. Inoculum size for weekly passage was determined in blood suspended in citrate saline or Alsever's, pH 7.2, using the Nile Blue method of Hillyer and Diggs (1964). Infected mice were bled out using cardiac puncture in syringes pre-loaded with sterile Alsever's (pH 7.2, 300 mOsm/liter). The infected blood was immediately added to tubes containing 40 ml Alsever's held in an ice bath. Sterile precautions were not maintained, although all solutions used were sterile. All subsequent manipulations were performed at 2 to 5 C. Cells were pelleted by centrifugation (1,000 g, 10 min), and supernatant fluid aspirated and discarded. Cells were then washed with 3 changes of Alsever's and resuspended in 2 vol Alsever's per vol packed cells. One milligram ADP per ml cell suspension was added with mixing, and the cells permitted to stand for 2 min. Cells were then passed through transfer pipettes packed with glass beads (3 g, 0.10 to 0.11 mm) overlaid with 3 g Whatman CFll powdered filter paper. This procedure gave nearly complete removal of leukocytes and platelets (Scheibel and Miller, 1969). After column treatment, the cells were washed twice with Alsever's, and the final volume adjusted to give a 50% suspension in Alsever's. Aliquots were removed for hemocytometric estimation of cell concentration (using Hayem's as diluent), and for preparation of films for determination of differential parasite counts. Wet mount preparations were made using cell suspensions loaded within Teflon seals (Carl Zeiss, Inc., New York) and overlaid with cover slips cleaned in nitric acid followed by rinses in distilled water, EDTA (1 mg/ml) and absolute ethanol. Wet mounts were observed using a Leitz Diavert microscope equipped with Smith differential interference contrast (DIC) optics. Cells were scored for the presence of discocytes, stomatocytes, and echinocytes (Brecher and Bessis, 1972), and were compared with preparations of cells from the same donor animal not exposed to the gradient material. Stractan II (food grade) was purchased from St. Regis Paper Co., Takoma, Wash. Solutions of Stractan were purified using exhaustive dialysis against distilled H20, followed by volume reduction using negative pressure dialysis and/or lyophilization. Densities of Stractan solutions were adjusted using refractometry, following initial determinations of refractive index vs. density of solutions determined gravimetrically (a linear relationship). Density determinations were performed at room temperature and corrected to 5 C. Adjustments of Stractan pH (7.4), and osmolarity (300 ? 10 mOsm/liter) were performed as outlined by Corash et al. (1974); osmolarity was measured using a freezing-point osmometer (Advanced instruments, Needham Heights, Mass.). Stock solutions of Stractan were sterilized using pressure filtration through a sterile 0.45 ,um filter, and stored at -20 C. Repetitive freezing and thawing of the material gave no evidence of deleterious effects, as measured by stable osmolarity and the absence of precipitate. Density distributions of parasitized cells were performed initially using the mixed phthalate ester method of Danon and Marikovsky (1964). Using specific gravity (sp gr) estimates of the different cell populations obtained with this method as a starting point, various methods were tested, employing both continuous and discontinuous gradients of Stractan solutions of varying densities. Pilot experiments were also used to empirically determine optimum run times/rotor speeds which would give equilibrium conditions for the non-pelleting fractions. The method of choice was found to be the use of a triple layered discontinuous preformed gradient. Cellulose nitrate tubes were loaded successively by careful layering with 1.5 ml each of 3 Stractan solutions, of densities 1.091, 1.081, and 1.043. Gradient building and cell additions were done in a cold room at 5 C. To the gradient was layered 0.5 ml washed cells in Alsever's. The loaded tubes were then centrifuged in the Beckman SW 50.1 rotor at 23,000 RPM (50,000 g) for 45 min at a rotor temperature of 5 C in a Beckman Model L ultracentrifuge. Following centrifugation, tubes were photographed and cells collected for subsequent analysis by puncture of the tube and aspiration cf the cells in each layer using a syringe.