Proc. Natl. Acad. Sci. USA
Vol. 75, No. 10, pp. 5081-5084, October 1978 Cell Biology
Isolation of malaria merozoites: Release of Plasmodium chabaudi merozoites from schizonts bound to immobilized concanavalin A (rodent malaria/concanavalin A-Sepharose/merozoite isolation)
PETER H. DAVID*, MARCEL HOMMEL*, JEAN-CLAUDE BENICHOUt, HARVEY A. EISEN*, AND Luiz H. PEREIRA DA SILVA* * Unit6 de Parasitologie Expeimentale, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France; and t Unite de Microscopie Electronique,
Institut Pasteur, Paris, France
Communicated by Andre Lwoff, August 8, 1978
P. chabaudi is known to have a fairly synchronous 24-hr asexual cycle in the mouse with a nocturnal peak of schizogony occurring at 0200 hr French Summer Time (FST) and leading to mature schizonts containing five or six merozoites (12). The schizogony was rendered diurnal by inverting the nycthemeral cycle of the mice. For this purpose, animals were kept in chambers artificially lit between 1800 and 0600 hr FST. After 10 days of such treatment, the animals were inoculated intraperitoneally with 107 parasitized erythrocytes. Schizogony then occurred between 1200 and 1800 hr FST, thus allowing diurnal experiments. Maintenance of the parasitic cycle was assured by intraperitoneal transfers of infected blood. Animals. O.F.1 male outbred mice (C.F.1 strain from Carworth Farms, bred by Iffa Credo, Fresnes, France), 4 to 6 weeks old, were used throughout the experiments. Cell Counts and Parasitemia. Erythrocyte counts were performed by using a Neubauer hemocytometer. Merozoite counts were performed by adding 1 vol of merozoite suspension to 1 vol of a chicken erythrocyte suspension of defined concentration and then examining a drop of this mixture by phase-contrast microscopy at a X400 magnification. The ratio merozoites/chicken erythrocytes was determined and merozoite concentration was calculated. Parasitemia was determined by microscopic examination of thin smears of tail blood fixed with methanol and stained with Giemsa. The number of parasitized cells per 500 erythrocytes was counted. The proportion of trophozoites and schizonts was determined in 200 infected cells. Concanavalin A-Sepharose Gel. Concanavalin A linked to beads of Sepharose 4B (Con A-Sepharose) was purchased from Pharmacia Fine Chemicals. The concanavalin A content was reported to be approximately 8 mg/ml of gel. Four washings were performed by suspending 1 vol of the gel in 3 vol of phosphate-buffered saline (pH 7.2) and centrifuging at 1000 X g for 5 min. The final pellet was suspended in 1 vol of phosphate-buffered saline at 370 and the gel was then ready for use. Blood Cell Suspension. When the level of parasitemia in a mouse had reached 30-60% and parasites were mainly schizonts, the animal was anesthetized with chloroform and bled by cardiac puncture. One milliliter of blood was collected into 30 units of heparin and diluted in 14 ml of phosphate-buffered saline at 370 containing 100 units of heparin. Medium. The medium used throughout the experiments was RPMI 1640 (GIBCO) buffered with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) and containing 5% fetal calf serum (Flow Laboratories). Infectivity of Merozoites. The method of Warhurst and Folwell (13) was used to compare in vivo infectivity of mero-
ABSTRACT The ability of concanavalin A to bind erythrocytes but not malarial parasites was used for the development of a method of merozoite isolation: cells from infected blood were allowed to bind to a column of concanavalin A linked to Sepharose beads and merozoites naturally released by maturation of the schizonts bound to the gel were collected. The principle of this method allows its application to several Plasmodium species. The kinetics of merozoite production and the quality of the preparations (purity, infectivity, and ultrastructural morphology) were investigated by using Plasmodium chabaudi.
The invasive merozoite liberated by rupture of the mature schizont during the erythrocyte cycle of the malarial parasite has been reported to have multiple interactions with the erythrocyte membrane and the host's immune system (1, 2). It is thus important to develop techniques for the large-scale production of merozoites required in biochemical and immunological studies. Methods based on the natural release of merozoites have been developed in human (3) and primate (4-6) malaria. In these techniques, concentrated schizonts are allowed to mature in vitro. When separated by agglutination of erythrocytes and immature parasites, the liberated merozoites are of limited viability (4). The use of a cell-sieve permits the isolation of a large number of intact and infective merozoites of Plasmodium knowlesi (6). However, the apparatus involved is relatively complex and this method has not been found useful for other plasmodial species, probably due to differences in size and deformability of erythrocytes, schizonts, and merozoites. In the case of rodent Plasmodium, free parasites have been obtained only by artificial lysis of infected erythrocytes. This type of preparation is always heavily contaminated by immature forms (for review, see ref. 7). In the present communication we describe a simple method for isolating merozoites by natural release. It is based on the ability of insolubilized concanavalin A to bind blood cells (8) but not merozoites (9) and should thus be applicable to various plasmodial species. The efficiency of this method and the morphology and infectivity of the merozoites produced were examined in the rodent model system Plasmodium chabaudi. MATERIALS AND METHODS Parasites. Strain IPPC1 of P. chabaudi Landau, 1965 (10) used throughout the experiments was obtained from the National Institute of Medical Research (Mill Hill, Great Britain); its identity had been confirmed by isoenzyme electrophoresis (11). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. § 1734 solely to indicate this fact.
Abbreviation: Con A-Sepharose, concanavalin A linked to beads of
Sepharose. 5081
5082
Cell Biology: David et al.
zoites to that of intraerythrocytic parasites. Briefly, three groups of mice were inoculated intravenously with 2 X 106, 2 X 105, and 2 X 104 merozoites, respectively, uncontaminated by any detectable erythrocytes. No more than 30 min passed between the beginning of the merozoite sample collection and the last mouse injection. Three other groups of six mice each were inoculated with equivalent numbers of intraerythrocytic trophozoites. Parasitemia was determined daily. For each mouse, the time taken to reach 2% parasitemia ("pre 2% period") was estimated by drawing a straight line on the logarithm of parasitemia-versus-time curve between two consecutive parasitemia values, one below and one above 2%. The mean "pre 2% periods" were then compared between groups. Electron Microscopy. Fixation was performed by adding 1 ml of 25% (wt/vol) glutaraldehyde (Sigma) to 9 ml of a merozoite suspension containing 3 X 106 organisms in medium. The samples were fixed for 6 hr at 40 and then centrifuged at 2000 X g for 5 min. The pellets were washed once in 0.1 M cacodylate buffer (pH 7.2) and the material was concentrated by centrifugation in agar as described for eukaryotic cells by Whitehouse et al. (14). Fixation was completed overnight in 0.2% osmium tetroxide in cacodylate buffer (pH 7.2) and then for 1 hr in 2% (wt/vol) uranyl acetate in water. After dehydration in an acetone series, the samples were embedded in Epon resin, cut with a Sorvall MT2 microtome, stained with 2% uranyl acetate for 2 min and lead citrate for 1 min, and examined with a Siemens 101 A electron microscope. RESULTS Isolation of Merozoites. In most experiments, 15 ml of Con A-Sepharose suspended in 15 ml of phosphate-buffered saline at 370 was poured and allowed to settle in a column made of a 20-ml plastic disposable syringe plugged with glass wool. The temperature in the column was maintained at 37° by means of a water jacket (a 50-ml plastic disposable syringe through which water was circulated from a water bath). A disc of filter paper was placed at the top of the Sepharose in order to avoid disturbance of the gel. Blood cell suspension was perfused through the column at a flow rate of 3 ml/min. The loaded column was rinsed with 20 ml of medium at a flow rate of 5 ml/min. Thereafter, the column was perfused with medium at a flow rate of 0.35
ml/min. Blood cells were counted in the original cell suspension and in the effluent from the column in order to calculate the effective number of erythrocytes bound to the Con A-Sepharose. Experiments performed with different quantities of Con ASepharose showed that 2.5-3.0 X 108 erythrocytes were bound per ml of gel. The size of the column could thus be chosen according to needs. Fig. 1 shows the kinetics of merozoite production in an average experiment in which a 15-ml column was used. The blood introduced into the column contained 55% infected erythrocytes (i.e., 51.5% trophozoites, 29.5% young schizonts with two nuclei, 15.5% schizonts with between two and five nuclei, and 3.5% mature schizonts with six nuclei). Of the 4.4 X 109 erythrocytes perfused through the column, 3.25 X 109 were retained by the gel, leading to the recovery of 5.75 X 108 merozoites during the 5-hi collection. In a series of 10 experiments, performed under similar conditions, the yield varied between 4 and 10 X 108 merozoites; the curve of merozoite production was always irregular. In view of the imperfect synchrony of P. chabaudi, it is not possible to make a straight-forward calculation of merozoite recovery. Because schizonts that are mature at the beginning of the experiment would only produce 3.6 X 108 merozoites,
Proc. Natl. Acad. Sci. USA 75 (1978) 20 X 16 co
Cl)
CO 12 CU
zo I-C
x
E4 Zo
Time, hr FIG. 1. Kinetics of P. chabaudi merozoite production: 3.25 X 109 erythrocytes were retained by a 15-nil column of Con A-Sepharose. FloW rate of medium was 0.35 mi/mmn. Samples were collected every 30 min and examined for merozoites (0), immature parasites (-), and erythrocytes (-).
one must assume that a certain degree of maturation occurs on the column during the experiment. Therefore, if one assumes that all parasites that are at the schizont stage at the time of bleeding are able to complete maturation in the column, the recovery was 10%. If only schizonts with more than two nuclei are considered to reach maturation, recovery was 30%o. Characteristics of the Merozoite Preparation. The main components of parasitized blood that can be expected to contaminate merozoite preparations based on natural release techniques are erythrocytes, erythrocyte membranes, immature parasites, residual bodies containing pigment, leukocytes, and platelets. As shown in Fig. 1, at the peak of merozoite production, erythrocyte contamination was very low to undetectable (less than 0.1%) and immature parasites never reached more than 6%. Leukocytes were not detected. Electron microscopy (Fig. 2) showed the presence of only few immature parasites, residual bodies, and membrane vesicles. Platelets and leukocytes were not observed. The morphology of the merozoites was well preserved; the apical complex and three sets of pellicular membranes were present as in merozoites of P. berghei and P. knnowllesl. No surface coat could be seen. Similar results were obtained with three different merozoite preparations. Fig. 3 shows that the "pre 2% periods" of mice that received merozoites and of mice that received trophozoites were of the same order of magnitude. If one assumes that 100% of trophozoites are viable at the time of inoculation and that a viable merozoite bears the same chance for future development as does a trophozoite, these results indicate a high infectivity of the merozoite preparation.
Cell Biology: David et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
5083
FIG. 2. Electron micrographs of P. chabaudi merozoite preparation. (a) General view. IP, immature parasites; RB, residual body; MV, membrane vesicle. Scale = 1 /Am. (b) Enlarged merozoite. Scale = 0.25 ,um.
DISCUSSION The simplicity of parasite maintenance in mice, the relative synchronicity of development, and the diurnal schizogony (which we obtained by inversion of the host's time cycle) make P. chabaudi an ideal model for exploring new methods of merozoite isolation. The great simplicity of the various components used in the method described-i.e., commercially available Con A-Sepharose, water bath, peristaltic pump, plastic syringes and tubing-contrasts with the relatively complex equipment required for the isolation of viable merozoites by the cell-sieve technique. Commercially available Con ASepharose does not have to be used. Concanavalin A is a lectin
that is easy to purify (15) and one can couple it to the Sepharose (16). Con A-Sepharose can be regenerated (17) but care must then be taken to control bacterial contamination; an average yield of merozoites was produced in one experiment with a column of regenerated Con A-Sepharose. When comparing the Con A-Sepharose technique applied to P. chabaudi with the cell-sieve technique applied to P. knowlesi, one should bear in mind that two very different parasites are involved: P. knowlesi is a primate malarial parasite readily maintained in vitro, producing 10-16 merozoites per schizont; in the cell-sieve, a suspension of 1 X 1011 pure schizonts, isolated by differential centrifugation, has been reported to produce 5 X 1010 merozoites (6). Such purification of schi-
Cell Biology: David et al.
5084
Proc. Natl. Acad. Sci. USA 75 (1978)
Electron microscopy shows the main morphological characteristics of well-preserved merozoites with the exception of the surface coat. One should nevertheless not assume the absence of a surface coat on P. chabaudi merozoites without further detailed microscopic studies. For example, photographic enhancement, used by Aikawa et al. (19) to help visualize the surface coat of P. knowlesi, could be applied. The contamination by erythrocyte membranes is low, but further treatment of merozoites may be needed if perfectly pure preparations are
10 CO cn
II
E 5
If
2 X14
2xlO5
2 x1o
Number of parasites injected FIG. 3. Time taken for parasitemia to reach 2% ("pre 2% period") in mice intravenously inoculated with P. chabaudi merozoites (0) or trophozoites (0). In each group, the arithmetic mean of the "pre 2% period" of six mice is expressed ±SD. zonts cannot be achieved with P. chabaudi; this parasite has only five or six merozoites per schizont, and optimal conditions for in vitro growth of rodent malarial parasites have yet to be determined (18). The fact that only a proportion of the schizonts bound to the column are able to complete maturation could explain why merozoite recovery did not exceed 10-30%, compared to the 50% obtained with the cell-sieve method using P. knowlesi. Loss of merozoites did not occur through direct binding of parasites to Con A-Sepharose, as shown in a parallel experiment in which merozoites were not retained when passed through a fresh Con A-Sepharose column, but invasion by merozoites of erythrocytes bound to the gel could be responsible for the loss of a certain number of parasites. Merozoite production has been scaled up through the use of several columns run in parallel; larger yields could also be produced by increasing the quantity of Con A-Sepharose per column. The irregular and widespread shape of the curve of merozoite production observed in all experiments reflects the imperfect synchrony of the parasite observed in vivo. Due to the imperfect culture techniques of rodent malarial parasites (11), infectivity of merozoites could only be estimated in vivo. Two different parasitic stages, the merozoite and the trophozoite, had to be compared. The fact that the "pre 2% period" of the merozoites was slightly inferior to that of trophozoites suggests that the viability of the merozoites was at least as satisfactory as the 20% viability obtained in cell-sieve preparations (6). The percentage of viable organisms cannot be determined.
required. Immobilized lectins offer a simple and adaptable method for merozoite isolation. The ability of membrane glycoproteins to bind to concanavalin A is shared by the blood cells of many species, including man and other primates. Results obtained in two preliminary experiments with P. knowlesi-infected rhesus erythrocytes were comparable to those obtained with P. chabaudi. This work was supported in part by the Institut National de la Sante et de la Recherche Scientifique, the Fondation pour la Recherche Medicale Franeaise, the Centre National de la Recherche Scientifique, and the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases. 1. Miller, L. H., Butcher, G. A. & Cohen, S. (1977) Bull. WHO 55, 157-162. 2. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1974) Nature (London) 252,311-313. 3. Mitchell, G. H., Richards, W. G. H., Butcher, G. A. & Cohen, S. (1977) Lancet i, 1335-1338. 4. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1973) Int. J. Parasitol. 3, 443-445. 5. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1975) Immunology 29,397-407. 6. Dennis, E. D., Mitchell, G. H., Butcher, G. A. & Cohen, S. (1975) Parasitology 71, 475-481. 7. Kreier, J. P. (1977) Bull. WHO 55,317-331. 8. Nicolson, G. L. (1974) Int. Rev. Cytol. 39,89-188. 9. Seed, T. M. & Kreier, J. P. (1976) Infect. Immun. 14, 13391347. 10. Landau, I. (1965) C. R. Hebd. Seances Acad. Sci. 260, 37583761. 11. Coombs, G. H. & Gutteridge, W. E. (1975) J. Protozool. 22, Part 4, 555-560. 12. Hawking, F., Gammage, K. & Worms, M. J. (1972) Parasitology 65, 189-201. 13. Warhurst, D. C. & Folwell, R. 0. (1968) Ann. Trop. Med. Parasitol. 62, 349-360. 14. Whitehouse, L. S., Benichou, J. C. & Ryter, A. (1977) Biol. Cell. 30, Part 2, 155-158. 15. Agrawal, B. B. L. & Goldstein, I. J. (1972) Methods Enzymol. 28, 314-316. 16. Edelman, G. M., Rutishauer, U. & Millette, C. F. (1971) Proc. Natl. Acad. Sci. USA 68,2153-2157. 17. Lloyd, K. 0. (1976) in Concanavalin A as a Tool, eds. Bittiger, H. & Schnebli, H. P. (Wiley, London,), pp. 323-331. 18. Trigg, P. I. (1976) Bull. WHO 53,399-406. 19. Aikawa, M., Miller, L. H., Johnson, J. & Rabbege, J. (1978) J. Cell Biol. 77, 72-82.
Vol. 75, No. 10, pp. 5081-5084, October 1978 Cell Biology
Isolation of malaria merozoites: Release of Plasmodium chabaudi merozoites from schizonts bound to immobilized concanavalin A (rodent malaria/concanavalin A-Sepharose/merozoite isolation)
PETER H. DAVID*, MARCEL HOMMEL*, JEAN-CLAUDE BENICHOUt, HARVEY A. EISEN*, AND Luiz H. PEREIRA DA SILVA* * Unit6 de Parasitologie Expeimentale, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France; and t Unite de Microscopie Electronique,
Institut Pasteur, Paris, France
Communicated by Andre Lwoff, August 8, 1978
P. chabaudi is known to have a fairly synchronous 24-hr asexual cycle in the mouse with a nocturnal peak of schizogony occurring at 0200 hr French Summer Time (FST) and leading to mature schizonts containing five or six merozoites (12). The schizogony was rendered diurnal by inverting the nycthemeral cycle of the mice. For this purpose, animals were kept in chambers artificially lit between 1800 and 0600 hr FST. After 10 days of such treatment, the animals were inoculated intraperitoneally with 107 parasitized erythrocytes. Schizogony then occurred between 1200 and 1800 hr FST, thus allowing diurnal experiments. Maintenance of the parasitic cycle was assured by intraperitoneal transfers of infected blood. Animals. O.F.1 male outbred mice (C.F.1 strain from Carworth Farms, bred by Iffa Credo, Fresnes, France), 4 to 6 weeks old, were used throughout the experiments. Cell Counts and Parasitemia. Erythrocyte counts were performed by using a Neubauer hemocytometer. Merozoite counts were performed by adding 1 vol of merozoite suspension to 1 vol of a chicken erythrocyte suspension of defined concentration and then examining a drop of this mixture by phase-contrast microscopy at a X400 magnification. The ratio merozoites/chicken erythrocytes was determined and merozoite concentration was calculated. Parasitemia was determined by microscopic examination of thin smears of tail blood fixed with methanol and stained with Giemsa. The number of parasitized cells per 500 erythrocytes was counted. The proportion of trophozoites and schizonts was determined in 200 infected cells. Concanavalin A-Sepharose Gel. Concanavalin A linked to beads of Sepharose 4B (Con A-Sepharose) was purchased from Pharmacia Fine Chemicals. The concanavalin A content was reported to be approximately 8 mg/ml of gel. Four washings were performed by suspending 1 vol of the gel in 3 vol of phosphate-buffered saline (pH 7.2) and centrifuging at 1000 X g for 5 min. The final pellet was suspended in 1 vol of phosphate-buffered saline at 370 and the gel was then ready for use. Blood Cell Suspension. When the level of parasitemia in a mouse had reached 30-60% and parasites were mainly schizonts, the animal was anesthetized with chloroform and bled by cardiac puncture. One milliliter of blood was collected into 30 units of heparin and diluted in 14 ml of phosphate-buffered saline at 370 containing 100 units of heparin. Medium. The medium used throughout the experiments was RPMI 1640 (GIBCO) buffered with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) and containing 5% fetal calf serum (Flow Laboratories). Infectivity of Merozoites. The method of Warhurst and Folwell (13) was used to compare in vivo infectivity of mero-
ABSTRACT The ability of concanavalin A to bind erythrocytes but not malarial parasites was used for the development of a method of merozoite isolation: cells from infected blood were allowed to bind to a column of concanavalin A linked to Sepharose beads and merozoites naturally released by maturation of the schizonts bound to the gel were collected. The principle of this method allows its application to several Plasmodium species. The kinetics of merozoite production and the quality of the preparations (purity, infectivity, and ultrastructural morphology) were investigated by using Plasmodium chabaudi.
The invasive merozoite liberated by rupture of the mature schizont during the erythrocyte cycle of the malarial parasite has been reported to have multiple interactions with the erythrocyte membrane and the host's immune system (1, 2). It is thus important to develop techniques for the large-scale production of merozoites required in biochemical and immunological studies. Methods based on the natural release of merozoites have been developed in human (3) and primate (4-6) malaria. In these techniques, concentrated schizonts are allowed to mature in vitro. When separated by agglutination of erythrocytes and immature parasites, the liberated merozoites are of limited viability (4). The use of a cell-sieve permits the isolation of a large number of intact and infective merozoites of Plasmodium knowlesi (6). However, the apparatus involved is relatively complex and this method has not been found useful for other plasmodial species, probably due to differences in size and deformability of erythrocytes, schizonts, and merozoites. In the case of rodent Plasmodium, free parasites have been obtained only by artificial lysis of infected erythrocytes. This type of preparation is always heavily contaminated by immature forms (for review, see ref. 7). In the present communication we describe a simple method for isolating merozoites by natural release. It is based on the ability of insolubilized concanavalin A to bind blood cells (8) but not merozoites (9) and should thus be applicable to various plasmodial species. The efficiency of this method and the morphology and infectivity of the merozoites produced were examined in the rodent model system Plasmodium chabaudi. MATERIALS AND METHODS Parasites. Strain IPPC1 of P. chabaudi Landau, 1965 (10) used throughout the experiments was obtained from the National Institute of Medical Research (Mill Hill, Great Britain); its identity had been confirmed by isoenzyme electrophoresis (11). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. § 1734 solely to indicate this fact.
Abbreviation: Con A-Sepharose, concanavalin A linked to beads of
Sepharose. 5081
5082
Cell Biology: David et al.
zoites to that of intraerythrocytic parasites. Briefly, three groups of mice were inoculated intravenously with 2 X 106, 2 X 105, and 2 X 104 merozoites, respectively, uncontaminated by any detectable erythrocytes. No more than 30 min passed between the beginning of the merozoite sample collection and the last mouse injection. Three other groups of six mice each were inoculated with equivalent numbers of intraerythrocytic trophozoites. Parasitemia was determined daily. For each mouse, the time taken to reach 2% parasitemia ("pre 2% period") was estimated by drawing a straight line on the logarithm of parasitemia-versus-time curve between two consecutive parasitemia values, one below and one above 2%. The mean "pre 2% periods" were then compared between groups. Electron Microscopy. Fixation was performed by adding 1 ml of 25% (wt/vol) glutaraldehyde (Sigma) to 9 ml of a merozoite suspension containing 3 X 106 organisms in medium. The samples were fixed for 6 hr at 40 and then centrifuged at 2000 X g for 5 min. The pellets were washed once in 0.1 M cacodylate buffer (pH 7.2) and the material was concentrated by centrifugation in agar as described for eukaryotic cells by Whitehouse et al. (14). Fixation was completed overnight in 0.2% osmium tetroxide in cacodylate buffer (pH 7.2) and then for 1 hr in 2% (wt/vol) uranyl acetate in water. After dehydration in an acetone series, the samples were embedded in Epon resin, cut with a Sorvall MT2 microtome, stained with 2% uranyl acetate for 2 min and lead citrate for 1 min, and examined with a Siemens 101 A electron microscope. RESULTS Isolation of Merozoites. In most experiments, 15 ml of Con A-Sepharose suspended in 15 ml of phosphate-buffered saline at 370 was poured and allowed to settle in a column made of a 20-ml plastic disposable syringe plugged with glass wool. The temperature in the column was maintained at 37° by means of a water jacket (a 50-ml plastic disposable syringe through which water was circulated from a water bath). A disc of filter paper was placed at the top of the Sepharose in order to avoid disturbance of the gel. Blood cell suspension was perfused through the column at a flow rate of 3 ml/min. The loaded column was rinsed with 20 ml of medium at a flow rate of 5 ml/min. Thereafter, the column was perfused with medium at a flow rate of 0.35
ml/min. Blood cells were counted in the original cell suspension and in the effluent from the column in order to calculate the effective number of erythrocytes bound to the Con A-Sepharose. Experiments performed with different quantities of Con ASepharose showed that 2.5-3.0 X 108 erythrocytes were bound per ml of gel. The size of the column could thus be chosen according to needs. Fig. 1 shows the kinetics of merozoite production in an average experiment in which a 15-ml column was used. The blood introduced into the column contained 55% infected erythrocytes (i.e., 51.5% trophozoites, 29.5% young schizonts with two nuclei, 15.5% schizonts with between two and five nuclei, and 3.5% mature schizonts with six nuclei). Of the 4.4 X 109 erythrocytes perfused through the column, 3.25 X 109 were retained by the gel, leading to the recovery of 5.75 X 108 merozoites during the 5-hi collection. In a series of 10 experiments, performed under similar conditions, the yield varied between 4 and 10 X 108 merozoites; the curve of merozoite production was always irregular. In view of the imperfect synchrony of P. chabaudi, it is not possible to make a straight-forward calculation of merozoite recovery. Because schizonts that are mature at the beginning of the experiment would only produce 3.6 X 108 merozoites,
Proc. Natl. Acad. Sci. USA 75 (1978) 20 X 16 co
Cl)
CO 12 CU
zo I-C
x
E4 Zo
Time, hr FIG. 1. Kinetics of P. chabaudi merozoite production: 3.25 X 109 erythrocytes were retained by a 15-nil column of Con A-Sepharose. FloW rate of medium was 0.35 mi/mmn. Samples were collected every 30 min and examined for merozoites (0), immature parasites (-), and erythrocytes (-).
one must assume that a certain degree of maturation occurs on the column during the experiment. Therefore, if one assumes that all parasites that are at the schizont stage at the time of bleeding are able to complete maturation in the column, the recovery was 10%. If only schizonts with more than two nuclei are considered to reach maturation, recovery was 30%o. Characteristics of the Merozoite Preparation. The main components of parasitized blood that can be expected to contaminate merozoite preparations based on natural release techniques are erythrocytes, erythrocyte membranes, immature parasites, residual bodies containing pigment, leukocytes, and platelets. As shown in Fig. 1, at the peak of merozoite production, erythrocyte contamination was very low to undetectable (less than 0.1%) and immature parasites never reached more than 6%. Leukocytes were not detected. Electron microscopy (Fig. 2) showed the presence of only few immature parasites, residual bodies, and membrane vesicles. Platelets and leukocytes were not observed. The morphology of the merozoites was well preserved; the apical complex and three sets of pellicular membranes were present as in merozoites of P. berghei and P. knnowllesl. No surface coat could be seen. Similar results were obtained with three different merozoite preparations. Fig. 3 shows that the "pre 2% periods" of mice that received merozoites and of mice that received trophozoites were of the same order of magnitude. If one assumes that 100% of trophozoites are viable at the time of inoculation and that a viable merozoite bears the same chance for future development as does a trophozoite, these results indicate a high infectivity of the merozoite preparation.
Cell Biology: David et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
5083
FIG. 2. Electron micrographs of P. chabaudi merozoite preparation. (a) General view. IP, immature parasites; RB, residual body; MV, membrane vesicle. Scale = 1 /Am. (b) Enlarged merozoite. Scale = 0.25 ,um.
DISCUSSION The simplicity of parasite maintenance in mice, the relative synchronicity of development, and the diurnal schizogony (which we obtained by inversion of the host's time cycle) make P. chabaudi an ideal model for exploring new methods of merozoite isolation. The great simplicity of the various components used in the method described-i.e., commercially available Con A-Sepharose, water bath, peristaltic pump, plastic syringes and tubing-contrasts with the relatively complex equipment required for the isolation of viable merozoites by the cell-sieve technique. Commercially available Con ASepharose does not have to be used. Concanavalin A is a lectin
that is easy to purify (15) and one can couple it to the Sepharose (16). Con A-Sepharose can be regenerated (17) but care must then be taken to control bacterial contamination; an average yield of merozoites was produced in one experiment with a column of regenerated Con A-Sepharose. When comparing the Con A-Sepharose technique applied to P. chabaudi with the cell-sieve technique applied to P. knowlesi, one should bear in mind that two very different parasites are involved: P. knowlesi is a primate malarial parasite readily maintained in vitro, producing 10-16 merozoites per schizont; in the cell-sieve, a suspension of 1 X 1011 pure schizonts, isolated by differential centrifugation, has been reported to produce 5 X 1010 merozoites (6). Such purification of schi-
Cell Biology: David et al.
5084
Proc. Natl. Acad. Sci. USA 75 (1978)
Electron microscopy shows the main morphological characteristics of well-preserved merozoites with the exception of the surface coat. One should nevertheless not assume the absence of a surface coat on P. chabaudi merozoites without further detailed microscopic studies. For example, photographic enhancement, used by Aikawa et al. (19) to help visualize the surface coat of P. knowlesi, could be applied. The contamination by erythrocyte membranes is low, but further treatment of merozoites may be needed if perfectly pure preparations are
10 CO cn
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2 X14
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2 x1o
Number of parasites injected FIG. 3. Time taken for parasitemia to reach 2% ("pre 2% period") in mice intravenously inoculated with P. chabaudi merozoites (0) or trophozoites (0). In each group, the arithmetic mean of the "pre 2% period" of six mice is expressed ±SD. zonts cannot be achieved with P. chabaudi; this parasite has only five or six merozoites per schizont, and optimal conditions for in vitro growth of rodent malarial parasites have yet to be determined (18). The fact that only a proportion of the schizonts bound to the column are able to complete maturation could explain why merozoite recovery did not exceed 10-30%, compared to the 50% obtained with the cell-sieve method using P. knowlesi. Loss of merozoites did not occur through direct binding of parasites to Con A-Sepharose, as shown in a parallel experiment in which merozoites were not retained when passed through a fresh Con A-Sepharose column, but invasion by merozoites of erythrocytes bound to the gel could be responsible for the loss of a certain number of parasites. Merozoite production has been scaled up through the use of several columns run in parallel; larger yields could also be produced by increasing the quantity of Con A-Sepharose per column. The irregular and widespread shape of the curve of merozoite production observed in all experiments reflects the imperfect synchrony of the parasite observed in vivo. Due to the imperfect culture techniques of rodent malarial parasites (11), infectivity of merozoites could only be estimated in vivo. Two different parasitic stages, the merozoite and the trophozoite, had to be compared. The fact that the "pre 2% period" of the merozoites was slightly inferior to that of trophozoites suggests that the viability of the merozoites was at least as satisfactory as the 20% viability obtained in cell-sieve preparations (6). The percentage of viable organisms cannot be determined.
required. Immobilized lectins offer a simple and adaptable method for merozoite isolation. The ability of membrane glycoproteins to bind to concanavalin A is shared by the blood cells of many species, including man and other primates. Results obtained in two preliminary experiments with P. knowlesi-infected rhesus erythrocytes were comparable to those obtained with P. chabaudi. This work was supported in part by the Institut National de la Sante et de la Recherche Scientifique, the Fondation pour la Recherche Medicale Franeaise, the Centre National de la Recherche Scientifique, and the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases. 1. Miller, L. H., Butcher, G. A. & Cohen, S. (1977) Bull. WHO 55, 157-162. 2. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1974) Nature (London) 252,311-313. 3. Mitchell, G. H., Richards, W. G. H., Butcher, G. A. & Cohen, S. (1977) Lancet i, 1335-1338. 4. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1973) Int. J. Parasitol. 3, 443-445. 5. Mitchell, G. H., Butcher, G. A. & Cohen, S. (1975) Immunology 29,397-407. 6. Dennis, E. D., Mitchell, G. H., Butcher, G. A. & Cohen, S. (1975) Parasitology 71, 475-481. 7. Kreier, J. P. (1977) Bull. WHO 55,317-331. 8. Nicolson, G. L. (1974) Int. Rev. Cytol. 39,89-188. 9. Seed, T. M. & Kreier, J. P. (1976) Infect. Immun. 14, 13391347. 10. Landau, I. (1965) C. R. Hebd. Seances Acad. Sci. 260, 37583761. 11. Coombs, G. H. & Gutteridge, W. E. (1975) J. Protozool. 22, Part 4, 555-560. 12. Hawking, F., Gammage, K. & Worms, M. J. (1972) Parasitology 65, 189-201. 13. Warhurst, D. C. & Folwell, R. 0. (1968) Ann. Trop. Med. Parasitol. 62, 349-360. 14. Whitehouse, L. S., Benichou, J. C. & Ryter, A. (1977) Biol. Cell. 30, Part 2, 155-158. 15. Agrawal, B. B. L. & Goldstein, I. J. (1972) Methods Enzymol. 28, 314-316. 16. Edelman, G. M., Rutishauer, U. & Millette, C. F. (1971) Proc. Natl. Acad. Sci. USA 68,2153-2157. 17. Lloyd, K. 0. (1976) in Concanavalin A as a Tool, eds. Bittiger, H. & Schnebli, H. P. (Wiley, London,), pp. 323-331. 18. Trigg, P. I. (1976) Bull. WHO 53,399-406. 19. Aikawa, M., Miller, L. H., Johnson, J. & Rabbege, J. (1978) J. Cell Biol. 77, 72-82.
