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Plasmodium berghei freed from host erythrocytes by a continuous-flow ultrasonic system
EXPERIMENT.4L
32, 239-243 (1972)
PARASITOLOGY
Plasmodium
berghei Freed Continuous-flow Richard
(Submitted
from Host Ultrasonic
B. Prior
and
for puh!ication.
Julius
Erythrocytes System’
by
a
P. Kreier
27 Dcc~mbcr.
1971)
PRIOR, R. B.. AXD KREIER, J. P., 1972. Pltrsnodi!tnr berghei freed from host crythrocytcs by a continuous-flow ultraronie system. Ezl)erimerctrrl Z’orGlology 32, 239-243 (1972). Ultrasound was used to free Plosn~otlirlm berghei from host erythrocytes. Debris formation, parasite damagr. and red cell-membrane entrallment of parasites were minimized by using a continuous-flow sy-stem of sonication. The free Iramsites were harvested by differential centrifugation, and low-power thin-section eleckon microscopy showed the effectiveness of the procedure. The free parasites were used aa antigens in complement-fixation tests, and they reacted wrll with sera from rats recovering from Plasmodizrm berghei infect,ion. INDEX
DESCRIPTOHS
Comp!ement-Fixation
: TJltrasound ; PEnsmodiun berghei; : Malaria ; Techniques ; Immumity.
Attempts to remove malarial parasites from their host erythrocytes by various physical and chemical techniques have resulted in incomplete separation of parasites from red cell membranes or extensive parasite damage (Cook et nl. 1969). Sonicat’ion of Plnsl,zodiu,,l-infected crythrocytcs re-leases parasites into the surrounding medium; but if these parasites are not removed from t’he sonication field, they are ihen exposed to the disrupt,& effects of the sonic energy and begin to disintegrate (Rutledge and Ward 1967). This study was mldertaken to develop a continuous-flow system of sonication in which each red cell would receive approximately equal exposure t,o sonic energy and in which the sonically released parasites would be removed from the disruptive sonic field promptly after rcle:N!. MATERIALS
AND
METHODS
‘This study was supported in part by Grant DSDA 17-72-C-2072 from the U.S. Army Medical Research and Development Command.
All
rights
239 @ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
electron ;
were collected by cardiac puncture using disodium ethylenediamine tetraacetate as the anticoagulant (1 mg/ml of whole blood). The erythrocytes were then washed three times in Alsever’s solution (pH 7.2) and resuspended in the same solution at a 10% concentration by volume as judged by high-speed hematocrit determination. The leukocytes were removed by passing the washed cell suspension through a tightly packed column of Whatman powdered filter paper (Fulton and Grant 1956). The sonication system consisted of a Bronwill Biosonik Model BP 1 Generator equipped with a standard 3/8-m. probe, a continuous-flow chamber, and a Harvard Model 1225 Rotary Peristaltic Pump. The continuous-flow chamber was constructed of 316 stainless steel and designed to reduce the eddying motions of the specimen within the chamber and to permit direct contact of the specimen with the transducer probe tip (Figs. 1 and 2j. Void volume of the sonication chamber was 0.2 cm’. The specimen was pumped through the chamber at a rate of 29.6 ml/min, and the power level was
Erythrocytes of rats acutely infected with P. berghel: (parasitemia over 40%)
Copyright
Microscof)y,
240
PRIOR
AND
TRANSDUCER
I
J
FIG. 1. Diagram of continuous-flow sonication chamber assembled and affixed to the transducer probe.
KREIER
adjusted to achieve approximately 80% red cell breakage. This power setting was determined by plotting percentage of hemolysis versus power setting (Fig. 3). Further increases in the power level settings did not increase the percentage of hemolysis significantly but resulted in increased parasite damage and debris formation. Heating of the specimen was not a problem due to the rapid flow of the precooled specimen. Freouency of sonication was 20,000 Hz. The sonicated specimen, which contained intact rat erythrocytes, LLghost” erythrocytes, free parasites, and host-cell membrane fractions, was subjected to differential centrifugation to harvest the free parasites. Initial centrifugation to remove the intact red cells was carried out at 275 g force. Height of specimen in the test tubo determined time of centrifugation and was calculated using the sedimentation rate for intact red cells. The sedimentation rate for washed rat erythrocytes in a capillary tube was 1.5 mm/hr at 1 g force. The free parasites were then sedimented at 600 g for 15 min and washed three times in Alsever’s solution (pH 7.2) to remove most of the ghost cells and host-cell membrane fractions. Some free plasmodia were lost in the initial
POWERDIAL SETTING (RELATIVE ULTRASONIC INTENSITY) bFIG. 2. Continuous-flow
the various dimensional uremenk are in inches.
2 ‘I4 ---j sonication chamber with measurements. All meas-
FIG. 3. Graph showing the rapid increase of hemolysis followed by a sharp reduction in the rate of increase of hemolysis with increasing ultrasonic intensity.
PlasnzO~~2r.?n-~LTRAsoNIC
FREEING
FROM
FIG. 4. Electron micrograph of a representative field of Plasmodium t.heir host erythrocytes by the continuous-flow sonication system.
ccl1 centrifugstion and in the washing ccss. ‘he free-parasite pellets were prepared
241
ERYTHROCYTES
berghei
freed from
for electron microscopy according to the procedure described by Cook et al. (1969), and thin sections were examined using a
242
PRIOR AND KREIER
TABLE I Complement-Fixation Titers of Normal Infected Rat Sera Using the FreeParasite Antigen
Group
No. in group
Daq’s postinoculation
Normal rats Infected rats
15 4
30
Infected rats
4
37
and
Titer’
NTh 8 32 32 64 16 16 16 32
n 50y0 hemolysis. b No titer at a 1:2 dilution of serum. Zeiss EM 9s electron microscope. The complement-fixation test procedure described by Kent and Fife (1963) was used to evaluate the free parasites as test antigens. Prior to use, the free parasites were lysed by three cycles of rapid freezing and thawing. Optimal dilution of the free-parasite lysate was determined in a block titration before use in the tests and the antigen was not anticomplementary at the employed dilution. RESULTS
Electron
Microscopy
Figure 4 is a low-power thin-section electron micrograph of a representative field of P. bergh.ei freed by continuous-flow sonication. Most of the parasites appear intact and are not surrounded by host-cell membranes. The parasites’ membranes and internal structure also appear to be well preserved. In addition to electron microscopic examination of the free parasites, smears of the free parasites were made and Giemsa stained. The free parasites showed normal staining characteristics. Serology The lysed free-parasite preparation utilized in the complement-fixation test re-
acted well with sera obtained from rats recovering from P. berghei infection. The rc sults obtained from two groups of recover ing rats at 30 and 37 days postinoculation are typical and are shown in Table I. The titers represent the highest dilution of antisera showing 50% hemolysis. Serum from no normal rats tested showed fixation while all rats previously infected with P. berghei showed fixation. The complement-fixation titers of rats with malaria varied from animal to animal and ranged from 8 to 64 in those animals tested. DISCUSSION
WC have shown by thin-section electron microscopy that a cavitating ultrasonic field can free malaria parasites from their host-cell membranes with little damage to the parasites and little debris formation. Initial serological tests using the free parasites show the antigens to react well wit)11 sera from rats recovering from primary P. berghei infection. All infected rats tested were shown to have complement-fixing antibodies in their sera while all normal rats tested were negative. The average titer was found to be slightly higher at 30 days postinoculation than at 37 days. Presently, we are studying the changes in complement-fixation titers during the course of the infection and the possible cross-reactions with other diseases. In addition, we are evaluating methods of lysing the free parasites for immunological studies as well as determining optimum conditions for storing this antigen. We believe this method of freeing malarial parasites provides unique opportunities for preparation of parasites freed of host cells for use in metabolic studies or as malarial antigens, Large quantities of infected blood can be processed quickly and economically. Minor modifications of the sonication system and centrifugation procedure may permit the isolation of other hemotrophic parasites.
Plasr~lOdizL~lz-r-LTRAsO~IC
ACKNOWLEDGMENTS We t,hank Dr. Robert Pfister and Owen Kindig for their assistance in the electron microscopy.
REFERENCES COOK, R. T.. AIKAWA. M., ROCK, R. C.. LITTLE. W.. AXD SPRINZ, H. 1969. The isolation and fractionation of Plasmodium knowlesi. Militnyq Medicine 134, 866-883. F~LTOS. J. D., AND GRANT, I’. T. 1956. The sulphur
FREEIKG FROM ERTTHROCYTES
24.3
requirements of the erythrocytic form of Plasmodium knowlesi. Biochemical Journal 63, 274-282. KENT, J. F., ASD FIFE, E. H. 1963. Precise standardization of reagents for complement fixation. 7’he American .lournnl of ‘I’ropicrrl Medicirle rend Hygiene 12, 103-116. RUTLEDGE, L. C.. AND WARD, R. A. 1967. Effects of ultrasound on Plnsmotlirtm gnllinnceiutn-infccted chick blood. Ezperimentd Pnrctsitology 20, 167-176.
32, 239-243 (1972)
PARASITOLOGY
Plasmodium
berghei Freed Continuous-flow Richard
(Submitted
from Host Ultrasonic
B. Prior
and
for puh!ication.
Julius
Erythrocytes System’
by
a
P. Kreier
27 Dcc~mbcr.
1971)
PRIOR, R. B.. AXD KREIER, J. P., 1972. Pltrsnodi!tnr berghei freed from host crythrocytcs by a continuous-flow ultraronie system. Ezl)erimerctrrl Z’orGlology 32, 239-243 (1972). Ultrasound was used to free Plosn~otlirlm berghei from host erythrocytes. Debris formation, parasite damagr. and red cell-membrane entrallment of parasites were minimized by using a continuous-flow sy-stem of sonication. The free Iramsites were harvested by differential centrifugation, and low-power thin-section eleckon microscopy showed the effectiveness of the procedure. The free parasites were used aa antigens in complement-fixation tests, and they reacted wrll with sera from rats recovering from Plasmodizrm berghei infect,ion. INDEX
DESCRIPTOHS
Comp!ement-Fixation
: TJltrasound ; PEnsmodiun berghei; : Malaria ; Techniques ; Immumity.
Attempts to remove malarial parasites from their host erythrocytes by various physical and chemical techniques have resulted in incomplete separation of parasites from red cell membranes or extensive parasite damage (Cook et nl. 1969). Sonicat’ion of Plnsl,zodiu,,l-infected crythrocytcs re-leases parasites into the surrounding medium; but if these parasites are not removed from t’he sonication field, they are ihen exposed to the disrupt,& effects of the sonic energy and begin to disintegrate (Rutledge and Ward 1967). This study was mldertaken to develop a continuous-flow system of sonication in which each red cell would receive approximately equal exposure t,o sonic energy and in which the sonically released parasites would be removed from the disruptive sonic field promptly after rcle:N!. MATERIALS
AND
METHODS
‘This study was supported in part by Grant DSDA 17-72-C-2072 from the U.S. Army Medical Research and Development Command.
All
rights
239 @ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
electron ;
were collected by cardiac puncture using disodium ethylenediamine tetraacetate as the anticoagulant (1 mg/ml of whole blood). The erythrocytes were then washed three times in Alsever’s solution (pH 7.2) and resuspended in the same solution at a 10% concentration by volume as judged by high-speed hematocrit determination. The leukocytes were removed by passing the washed cell suspension through a tightly packed column of Whatman powdered filter paper (Fulton and Grant 1956). The sonication system consisted of a Bronwill Biosonik Model BP 1 Generator equipped with a standard 3/8-m. probe, a continuous-flow chamber, and a Harvard Model 1225 Rotary Peristaltic Pump. The continuous-flow chamber was constructed of 316 stainless steel and designed to reduce the eddying motions of the specimen within the chamber and to permit direct contact of the specimen with the transducer probe tip (Figs. 1 and 2j. Void volume of the sonication chamber was 0.2 cm’. The specimen was pumped through the chamber at a rate of 29.6 ml/min, and the power level was
Erythrocytes of rats acutely infected with P. berghel: (parasitemia over 40%)
Copyright
Microscof)y,
240
PRIOR
AND
TRANSDUCER
I
J
FIG. 1. Diagram of continuous-flow sonication chamber assembled and affixed to the transducer probe.
KREIER
adjusted to achieve approximately 80% red cell breakage. This power setting was determined by plotting percentage of hemolysis versus power setting (Fig. 3). Further increases in the power level settings did not increase the percentage of hemolysis significantly but resulted in increased parasite damage and debris formation. Heating of the specimen was not a problem due to the rapid flow of the precooled specimen. Freouency of sonication was 20,000 Hz. The sonicated specimen, which contained intact rat erythrocytes, LLghost” erythrocytes, free parasites, and host-cell membrane fractions, was subjected to differential centrifugation to harvest the free parasites. Initial centrifugation to remove the intact red cells was carried out at 275 g force. Height of specimen in the test tubo determined time of centrifugation and was calculated using the sedimentation rate for intact red cells. The sedimentation rate for washed rat erythrocytes in a capillary tube was 1.5 mm/hr at 1 g force. The free parasites were then sedimented at 600 g for 15 min and washed three times in Alsever’s solution (pH 7.2) to remove most of the ghost cells and host-cell membrane fractions. Some free plasmodia were lost in the initial
POWERDIAL SETTING (RELATIVE ULTRASONIC INTENSITY) bFIG. 2. Continuous-flow
the various dimensional uremenk are in inches.
2 ‘I4 ---j sonication chamber with measurements. All meas-
FIG. 3. Graph showing the rapid increase of hemolysis followed by a sharp reduction in the rate of increase of hemolysis with increasing ultrasonic intensity.
PlasnzO~~2r.?n-~LTRAsoNIC
FREEING
FROM
FIG. 4. Electron micrograph of a representative field of Plasmodium t.heir host erythrocytes by the continuous-flow sonication system.
ccl1 centrifugstion and in the washing ccss. ‘he free-parasite pellets were prepared
241
ERYTHROCYTES
berghei
freed from
for electron microscopy according to the procedure described by Cook et al. (1969), and thin sections were examined using a
242
PRIOR AND KREIER
TABLE I Complement-Fixation Titers of Normal Infected Rat Sera Using the FreeParasite Antigen
Group
No. in group
Daq’s postinoculation
Normal rats Infected rats
15 4
30
Infected rats
4
37
and
Titer’
NTh 8 32 32 64 16 16 16 32
n 50y0 hemolysis. b No titer at a 1:2 dilution of serum. Zeiss EM 9s electron microscope. The complement-fixation test procedure described by Kent and Fife (1963) was used to evaluate the free parasites as test antigens. Prior to use, the free parasites were lysed by three cycles of rapid freezing and thawing. Optimal dilution of the free-parasite lysate was determined in a block titration before use in the tests and the antigen was not anticomplementary at the employed dilution. RESULTS
Electron
Microscopy
Figure 4 is a low-power thin-section electron micrograph of a representative field of P. bergh.ei freed by continuous-flow sonication. Most of the parasites appear intact and are not surrounded by host-cell membranes. The parasites’ membranes and internal structure also appear to be well preserved. In addition to electron microscopic examination of the free parasites, smears of the free parasites were made and Giemsa stained. The free parasites showed normal staining characteristics. Serology The lysed free-parasite preparation utilized in the complement-fixation test re-
acted well with sera obtained from rats recovering from P. berghei infection. The rc sults obtained from two groups of recover ing rats at 30 and 37 days postinoculation are typical and are shown in Table I. The titers represent the highest dilution of antisera showing 50% hemolysis. Serum from no normal rats tested showed fixation while all rats previously infected with P. berghei showed fixation. The complement-fixation titers of rats with malaria varied from animal to animal and ranged from 8 to 64 in those animals tested. DISCUSSION
WC have shown by thin-section electron microscopy that a cavitating ultrasonic field can free malaria parasites from their host-cell membranes with little damage to the parasites and little debris formation. Initial serological tests using the free parasites show the antigens to react well wit)11 sera from rats recovering from primary P. berghei infection. All infected rats tested were shown to have complement-fixing antibodies in their sera while all normal rats tested were negative. The average titer was found to be slightly higher at 30 days postinoculation than at 37 days. Presently, we are studying the changes in complement-fixation titers during the course of the infection and the possible cross-reactions with other diseases. In addition, we are evaluating methods of lysing the free parasites for immunological studies as well as determining optimum conditions for storing this antigen. We believe this method of freeing malarial parasites provides unique opportunities for preparation of parasites freed of host cells for use in metabolic studies or as malarial antigens, Large quantities of infected blood can be processed quickly and economically. Minor modifications of the sonication system and centrifugation procedure may permit the isolation of other hemotrophic parasites.
Plasr~lOdizL~lz-r-LTRAsO~IC
ACKNOWLEDGMENTS We t,hank Dr. Robert Pfister and Owen Kindig for their assistance in the electron microscopy.
REFERENCES COOK, R. T.. AIKAWA. M., ROCK, R. C.. LITTLE. W.. AXD SPRINZ, H. 1969. The isolation and fractionation of Plasmodium knowlesi. Militnyq Medicine 134, 866-883. F~LTOS. J. D., AND GRANT, I’. T. 1956. The sulphur
FREEIKG FROM ERTTHROCYTES
24.3
requirements of the erythrocytic form of Plasmodium knowlesi. Biochemical Journal 63, 274-282. KENT, J. F., ASD FIFE, E. H. 1963. Precise standardization of reagents for complement fixation. 7’he American .lournnl of ‘I’ropicrrl Medicirle rend Hygiene 12, 103-116. RUTLEDGE, L. C.. AND WARD, R. A. 1967. Effects of ultrasound on Plnsmotlirtm gnllinnceiutn-infccted chick blood. Ezperimentd Pnrctsitology 20, 167-176.