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Schistosoma mansoni: Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll
EXPERIMENTAL
PARASITOLOGY
53,39-44(1982)
Schistosoma mansoni: Rapid Isolation and Purification of Schistosomula of Different Developmental Stages by Centrifugation on Discontinuous Density Gradients of Percoll JANIS K. LAZDINS,’ Departments
MARSHA J. STEIN, JOHN R. DAVID,
AND ALAN SHER~
of Medicine and Pathology, Harvard Medical School and the Robert the Brigham and Women’s Hospital, Boston, Massachusetts 02115,
(Accepted for publication LAZDINS,J.K.,STEIN,M.J.,DAVID,J.R.,
B. Brigham U.S.A.
Division
of
8 December 1980) AND SHER,A.~~~~.
Schistosoma
mansoni:
Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll. Experimental Parasitology 53, 39-44. Step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll) were used to isolate and purify early development stages of Schistosoma mansoni (cercariae, skin stage, and 5-day-old schistosomula). With this method, mechanically transformed schistosomula can be isolated in higher purity and yield than that obtained with conventional procedures. In addition, use of the method revealed that schistosomula undergo a dramatic change in density during the first hours after transformation from cercariae. In other experiments, Sday-old schistosomula were effectively purified from contaminating lung tissue by means of the Percoll gradient procedure. After purification on Percoll, schistosomula display no evidence of damage when examined by light microscopy and no loss in viability as judged by recovery of adult worms from mice. INDEX DESCRIPTORS: Schistosoma mansoni; Trematoda; Blood Fluke; Percoll; Density gradient centrifugation; Mouse, skin, lung; Differentiation; Purification.
INTRODUCTION
entiation as well as the difference in density between worms and host tissue. Commercially available polyvinylpyrolidone-coated colloidal silica particles (Percoll) are used to produce stable density gradients of large capacity. Schistosomula can be purified in high yield on these gradients and with no loss in viability. Using this method we have revealed a striking change in the density of schistosomula occurring during the first few hours after their transformation from cercariae.
Recently there has been considerable interest in the biological and biochemical properties of the schistosomulum because of its probable importance as a major target of host immunological attack (Smithers and Terry 1976). A limitation in pursuing this work has been the lack of effective techniques for separating different larval schistosome stages from each other, as well as from contaminating host material. In this paper we describe a method for the purification of schistosomula which takes advantage of the changes in densities that the parasite exhibits during its differ-
MATERIALS
AND
METHODS
Parasites. The Puerto Rican strain of Schistosoma mansoni is maintained in our laboratory by passage through Biomphalaria gfabrata snails and outbred CF,
1 Present address: Laboratory of Immunobiology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A. z Present address: Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, Bethesda, MD 20205, U.S.A.
mice (Charles River Breeding Laboratories, Wilmington, MA, USA). In the studies to be reported, schistosomula were prepared 39
0014-4894/82/010039-06$02.00/O Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
40
LAZDINS
from cercariae using two methods. The first method involves penetration of cercariae through rat skin and recovery of schistosomula 3-4 hr later as previously described (Stirewalt et al. 1966; Clegg and Smithers 1972). The second method originally reported by Ramalho-Pinto et al. (1974) involves mechanical transformation of cercariae to schistosomula induced by shearing in a Vortex mixer. After detachment by this procedure, cercarial bodies were separated from trails on Percoll gradients as outlined in Fig. 1. Five-day-old schistosomula were obtained by the following procedure. CBA mice (Jackson Laboratories, Bar Harbor, ME, USA) were injected intravenously (iv) with 2 x 10” mechanically transformed schistosomula or subcutaneously with 3 x lo3 cercariae. Five days later, the mice were sacrificed, the lungs removed and chopped, and schis[
FIG.
for the preparation schistosomula method of Ramalho-Pinto et al. (1974) with the newer technique employing bodies from tails by centrifugation on Percoll gradients. Schistosoma
1. Scheme
mansoni
of mechanical employing the as compared separation of discontinuous
ET AL.
tosomula recovered from lung fragments as described previously (Sher et al. 1974, 1978). In several experiments, lung schistosomula preparations were further purified by passage through nylon-wool columns followed by two cycles of sedimentation (20 min each) at Ig in 15ml conical centrifuge tubes (Butterworth et al. 1979). The purification of 5-day-old schistosomula using Percoll gradients is described under Results. Preparation of density gradients. Percoll (polyvinylpyrolidone-coated colloidal silica particles, starting density 1.135 g/ml) was purchased from Pharmacia Fine Chemicals (Piscataway, NJ, USA) (Pertoft et al. 1978). Isotonic solutions of Percoll of different densities were prepared by diluting the stock suspension with the appropriate volumes of 10x concentrated minimal essential medium (MEM) (Microbiological Associates, Walkersville, MD, USA) and distilled water. The solutions were then buffered with 1 M Hepes (Microbiological Associates) to a final concentration of 25 mM and the pH adjusted to 7.0-7.2 with 1 N NaOH. The densities (g/ml) calculated from manufacturers specifications were as follows: 1.044 (30%), 1.058 (40%), 1.068 (50%), 1.085 (60%), and 1.099 (70%). These solutions were stored at 4 C before use. To achieve sharp interfaces, 10 ml of each of these solutions was layered from the bottom of 50-ml plastic conical centrifuge tubes (Bioquest, BBL, Falcon Products, Becton-Dickinson, Cockeysville, MD, USA) using a lPgauge, 5-in.-long needle placed in the tube. This needle was connected to a lo-ml syringe via a three-way stopcock that allowed regulation of the flow rate of the Percoll solutions. The samples to be fractionated were layered at the top of the gradient in a volume of 5 ml of medium. The separation was achieved by low-speed centrifugation (35Og) for 10 min at 5 C. Five-milliliter samples were then collected from the bottom of the tube after perforation with a heated 16-gauge needle. The collected fractions were diluted in IO-vol of
Schistosoma
mansoni:
RAPID
MEM and the schistosomula pelleted by centrifugation. The samples were washed two additional times before further use. No attempt was made to determine the density of the collected fractions and they are designated according to the estimated percentage of Percoll which they contain. Assay of viability. The viability of schistosomula preparations were evaluated by the following criteria. (1) Direct microscopic examination of parasite motility and activity of flame cells. Parasites were scored as dead if they were immotile, nontranslucent, and had become granular and opaque in appearance. (2) Dye exclusion. Microscope slides were precoated with a drop of 0.1% toluidine blue solution in methanol, which was allowed to dry. A sample was placed on the slide and examined by light microscopy (Butterworth et al. 1979). Parasites were scored as dead when they were observed to take up large amounts of dye. (3) In vivo recovery. Precounted quantities of schistosomula (100-200) were injected iv into the tail veins of CBA/J mice. Six weeks later the animals were perfused (Smithers and Terry 1965) and the recovery of adult worms was determined.
ISOLATION
OF
SCHISTOSOMULA
41
were centrifuged in a discontinuous Percoll density gradient. A clear-cut separation of dead from live worms was observed (Fig. 2). The living organisms (which by morphology and dye exclusion were 100% viable) were found in the 50 and 60% interfaces. In contrast, the dead organisms were observed to band in the 30 and 40% interfaces. A similar separation was achieved when a mixture of live and killed (by exposure to 1 m&f N-maleimide) mechanically prepared schistosomula was applied to the gradient (data not shown). As can be seen in Fig. 2, the method fails to separate cercariae present in the preparation from living schistosomula. Separation of Mechanically Detached Cercarial Bodies from Tails Bodies and tails were prepared from lo5 cercariae by mechanical disruption, resuspended in 5 ml of MEM, and loaded on the top of individual gradients. After centrifugation, ten 5-ml fractions were collected and the number of bodies and tails in each fraction was enumerated. Results from a typical experiment are shown in Fig. 3. It can be seen that 90% of the cercarial bodies were found at the bottom of the tube in a
RESULTS
Purification of Schistosomula by Skin Penetration
Prepared
When Schistosoma mansoni cercariae are transformed to schistosomula by the skin penetration method, the preparations are usually contaminated with dead organisms (5-20%). This contamination becomes critical in experiments where the schistosomula are to be used as targets in in vitro killing experiments. Previously it was noticed that dead organisms are more difficult to pellet by centrifugation. This observation suggests that significant changes in density occur when schistosomula are killed. In order to test this hypothesis, preparations of skin-stage schistosomula highly contaminated with dead organisms
% PERCOU
2. Separation by centrifugation on Percoll of live from dead Schisrosoma mansoni schistosomula transformed from cercariae by skin penetration. A preparation of schistosomula consisting of 6% living schistosomula, 20% dead schistosomula, and 11% cercariae was loaded onto Percoll gradients. After centrifugation, 5-ml fractions were collected and the numbers of living and dead schistosomula (evaluated by morphologic examination and dye exclusion) and cercariae present in each fraction were counted. FIG.
42
LAZDINS
3i? ap
Cercariol
Bodies
Cercarial
Tails
50
25 0 30
40
50
60
70
% PERcoLL
FIG. 3. Separation of Schistosoma mansoni cercarial bodies from tails by centrifugation on Percoll. A population of freshly shed cercariae (105) was mechanically disrupted into bodies and tails and the preparation separated on Percoll as outlined in Fig. 1.
ET AL.
and 60% fractions and were highly contaminated with tails. This observation led us to examine the change in density undergone by cercarial bodies during their transformation to schistosomula. An example of an experiment demonstrating this density shift is shown in Fig. 4. It can be seen that after 150 min of incubation, the density distribution is very similar to that observed when schistosomula produced by skin penetration are separated on the gradient (Fig. 2). In addition, morphological examination (by Normarski interference microscopy) revealed that the majority of the larvae recovered from the gradient after incubation had lost their glycocalyx as well as the contents of their acetabular glands. No attempt was made to quantitate this relationship.
fraction contaminated with less than 1% tails. The few intact cercariae remaining after mechanical transformation were also found in this fraction. In contrast, most tails of j-Day-Old Schistosomula were found at the interface of the 40 and Purification from Contaminating Lung Tissue 50% Percoll fractions. The fraction conand Cells taining the cercarial bodies was washed twice in MEM and incubated for 2 hr at 37 Schistosomula were recovered from the C. Schistosomula prepared in this manner lungs of mice 5 days after intravenous inwere found to be 100% viable as judged by jection according to standard procedures morphologic examination and dye exclusion and were obtained in yields greater than 90% as compared with the yields of 30-40% obtained by means of the conventional procedure of Ramalho-Pinto et al. (1974). In later experiments it was found that the above protocol for the purification of mechanical schistosomula could be simplified by fractionating the sheared cercariae on single cushion of 70% Percoll (50,000 parasites/10 ml). After centrifugation (10 min 35Og), the cercarial tails were found at the top of the cushion and the bodies in a pellet at the bottom of the tube. The yields of schistosomula using this modified proce30 40 50 60 70 dure were 90% or greater and the purity % PERCOU excellent (better than 95%). FIG. 4. Density change in Schistosoma mansoni from cercarial bodies into If the suspension of tails and bodies was larvae during transformation mature schistosomula. Cercarial bodies (105) were infirst incubated to achieve the transformacubated in Earles/lactalbumin medium at 37 C and at tion to schistosomula, and then placed on the intervals indicated aliquots (2 x lo4 organisms) gradients, it was observed that the schis- were loaded onto individual Percoll gradients and the tosomula did not pellet to the bottom but mobility of the larvae in the gradients was analyzed instead were found in the interface of the 50 after centrifugation (10 min).
Schistosoma
t?lanSOtZi:
RAPID
(Sher et al. 1974). The worm preparations from 10 mice were resuspended in a total of 5 ml of MEM and placed at the top of the gradient, centrifuged, and processed. Most of the 5-day-old schistosomula (70%) were observed to band in the 50% Percoll fraction (Fig. 5). In contrast, the large fragments of lung tissue remained in the 30% fraction and most single cells were scattered in the 30 and 40% fractions. The viability of the lung stage schistosomula purified on Percoll gradients was found to be greater than 95% as determined by morphological examination and dye exclusion. This finding was corroborated by injecting mice with the isolated schistosomula and assaying the recovery of adult worms 6 weeks later. The results are presented in Table I. No significant differences in recovery were observed from animals that had received 5-day-old schistosomula prepared by the traditional method of nylon-wool liltration and from animals which received schistosomula purified on density gradients (Experiments 1 and 3) and indeed in one experiment (No. 2) the recovery from animals that had received 5-day-old schistosomula prepared by density gradient centrifugation was significantly higher than the recovery from animals that had received worms purified by the nylon-wool technique. DISCUSSION
Gradients of colloidal silica have been used successfully to isolate cells and sub-
ISOLATION
OF
SCHISTOSOMULA
30
40
50
60
70
% PERCOU
FIG. 5. Separation of S-day-old Schistosoma mansoni schistosomula from contaminating host tissue by centrifugation on Percoll. The inserts indicate the distribution of free lung cells or lung fragments (LF) on the gradient.
cellular particles of various types including liver cells, HeLa cells, viruses, lysozomes, mitochondria, etc. (Pertoft and Laurent 1977). Colloidal silica particles coated with polyvinylpyrolidone are nontoxic and offer advantages over conventional materials such as sucrose of metrizamide because of their low viscosity, low osmolality, and impermeability to biological membranes. In this paper we have described the use of this material to form discontinuous gradients which are employed to purify Schistosoma mansoni schistosomula of different developmental stages. Our data indicate that schistosomula isolated by centrifugation on Percoll exhibit no loss in viability, as judged by morphologic examination and dye exclusion and by their capacity to develop into adult worms after injection in mice (Table I). Furthermore, with most of the currently available methods for the iso-
TABLE I Infectivity of 5-Day-Old Schistosoma mansoni Schistosomula, Purified by Percoll Gradient Centrifugation Percent recovery of adult worms from mice injected with: Experiment 1 2 3
Percollpurified larvae
Nylon-wool purified larvae
P value
41 k 4.7 (5, 65)O 56 ?I 2.9 (7, 200) 25 k 1.7 (8, 170)
55 k 5.7 (5, 106) 41 ” 3.3 (7, 200) 25 k 1.3 (8, 170)
0.085 (NS)* 0.005 0.909 (NS)
a Results are presented as means k SEM. The numbers in parentheses refer respectively, to the number of mice perfused in each experiment and the average number of larvae injected per mouse. * Not statistically significant.
44
LAZDINS
lation and purification of schistosomula, yield is sacrificed for the sake of purity. With the use of the Percoll centrifugation technique, we have achieved high purity with yields often greater than 90%. The experiments reported here (Fig. 4) reveal that when cercariae are transformed in vitro to schistosomula, there is a simultaneous shift in the density of the cercarial bodies such that by 150 min of incubation the density of the organisms is indistinguishable from that of skin-stage schistosomula. Acetabular gland secretion, the loss of the cercarial glycocalyx, and changes in membrane permeability are known to occur during the same period (Stirewalt and Uy 1969; Gazzinelli et al. 1973) and probably underlie the density transition observed. For this reason, parasite density, as determined by Percoll centrifugation, may prove to be a useful analytical parameter of irt vitro transformation. A final application of the Percoll separation technique described in the present study was the use of the method to purify schistosomula from contaminating host tissue. Previous attempts to isolate 5-day-old parasites free of cells and tissue fragments by means of sedimentation and filtration through nylon-wool columns (Butterworth et al. 1979) have met with only partial success. By means of density centrifugation on Percoll, it is now possible to isolate 5-dayold schistosomula in sufficient purity and yield to allow biochemical analysis. For instance, it should now be feasible to chemically characterize the parasite antigens as well as host molecules associated with the larval surface, a problem which until now has been impractical to pursue because of the contamination of schistosomulum preparations with host tissue. ACKNOWLEDGMENTS This work was supported by grants from the Rockefeller Foundation, The Clark Foundation, and the U.S. National Institute of Allergy and Infectious Diseases. We thank John Samuelson for his helpful discussions and suggestions and Christine Sleiman for her assistance in the preparation of this manuscript.
ET AL. REFERENCES A. E., VADAS, M. A., MARTZ, E., A. 1979. Cytolytic T lymphocytes recognize alloantigens on schistosomula of Schistosoma mansoni, but fail to induce damage. Journal of fmmanology 122, 1314- 1321. CLEGG, J. A., AND SMITHERS, S. R. 1972. The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International Journal for Parasitology 2, 79-98. BUTTERWORTH, AND SHER,
GAZZINELLI,
G.,
OLIVEIRA,
C. C.,
FIGUEIRREDO,
L. H., COELHO, P. M. Z., AND 1973. Schistosoma mansoni: Biochemical evidence for morphogenetic change of cercaria to schistosomule. Experimental Parasitology 34, 181-188. PERTOFT. H., AND LAURENT, T. C. 1977. Isopycnic separation of cells and organelles by centrifugation in modified silica gradient. In “Methods of Cell Separation” (N. Catsimpoolas, ed.), pp. 25-65, Plenum, New York. PERTOFT, H., LAURENT, T. C., LAAS, T., AND KAGEDAL, L. 1978. Density gradients prepared from colloidal silica particles coated by polyvinylpyrolidone (Percoll). Analytical Biochemistry 88, 271-282. RAMALHO-PINTO, F. J., GAZZINELLI, G., HOWELLS, R. E., MOTA-SANTOS, T. A., FIGUIEREDO, E. A., AND PELLEGRINO, J. 1974. Schistosoma mansoni: Defined system for stepwise transformation of cercaria to schistosomule in vitro. Experimental Parasitology 36, 360-372. E. A., PEREIRA, PELLEGRINO, J.
SHER,
F. A.,
HALL,
B. F.,
AND
VADAS,
M.
A.
1978.
Acquisition of murine major histocompatibility complex gene products by schistosomula of Schistosoma mansoni. Journal of Experimental Medicine 148, 46-57. F. A., MACKENZIE, P., AND SMITHERS, S. R. 1974. Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the laboratory mouse. Journal of Infectious Diseases 130, 626-633.
SHER,
SMITHERS,
S. R.,
AND
TERRY,
tion of laboratory hosts with soma mansoni and the recovery Parasitology 55, 695-700.
R. J. 1965. The infeccercariae of Schistoof the adult worms.
AND TERRY, R. J. 1976. The imof schistosomiasis. Advances in Para14, 399-422. STIREWALT, M. A., MINNICK, D. R. R., AND FREGEAU, W. A. 1966. Definition and collection in quantity of schistosomules ofschistosoma mansoni. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 352-360. STIREWALT, M. A., AND UY, A. 1969. Schistosoma mansoni: Cercarial penetration and schistosomule collection in an in vitro system. Experimental Parasitology 26, 17-28. SMITHERS,
munology sitology
S. R.,
PARASITOLOGY
53,39-44(1982)
Schistosoma mansoni: Rapid Isolation and Purification of Schistosomula of Different Developmental Stages by Centrifugation on Discontinuous Density Gradients of Percoll JANIS K. LAZDINS,’ Departments
MARSHA J. STEIN, JOHN R. DAVID,
AND ALAN SHER~
of Medicine and Pathology, Harvard Medical School and the Robert the Brigham and Women’s Hospital, Boston, Massachusetts 02115,
(Accepted for publication LAZDINS,J.K.,STEIN,M.J.,DAVID,J.R.,
B. Brigham U.S.A.
Division
of
8 December 1980) AND SHER,A.~~~~.
Schistosoma
mansoni:
Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll. Experimental Parasitology 53, 39-44. Step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll) were used to isolate and purify early development stages of Schistosoma mansoni (cercariae, skin stage, and 5-day-old schistosomula). With this method, mechanically transformed schistosomula can be isolated in higher purity and yield than that obtained with conventional procedures. In addition, use of the method revealed that schistosomula undergo a dramatic change in density during the first hours after transformation from cercariae. In other experiments, Sday-old schistosomula were effectively purified from contaminating lung tissue by means of the Percoll gradient procedure. After purification on Percoll, schistosomula display no evidence of damage when examined by light microscopy and no loss in viability as judged by recovery of adult worms from mice. INDEX DESCRIPTORS: Schistosoma mansoni; Trematoda; Blood Fluke; Percoll; Density gradient centrifugation; Mouse, skin, lung; Differentiation; Purification.
INTRODUCTION
entiation as well as the difference in density between worms and host tissue. Commercially available polyvinylpyrolidone-coated colloidal silica particles (Percoll) are used to produce stable density gradients of large capacity. Schistosomula can be purified in high yield on these gradients and with no loss in viability. Using this method we have revealed a striking change in the density of schistosomula occurring during the first few hours after their transformation from cercariae.
Recently there has been considerable interest in the biological and biochemical properties of the schistosomulum because of its probable importance as a major target of host immunological attack (Smithers and Terry 1976). A limitation in pursuing this work has been the lack of effective techniques for separating different larval schistosome stages from each other, as well as from contaminating host material. In this paper we describe a method for the purification of schistosomula which takes advantage of the changes in densities that the parasite exhibits during its differ-
MATERIALS
AND
METHODS
Parasites. The Puerto Rican strain of Schistosoma mansoni is maintained in our laboratory by passage through Biomphalaria gfabrata snails and outbred CF,
1 Present address: Laboratory of Immunobiology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A. z Present address: Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, Bethesda, MD 20205, U.S.A.
mice (Charles River Breeding Laboratories, Wilmington, MA, USA). In the studies to be reported, schistosomula were prepared 39
0014-4894/82/010039-06$02.00/O Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
40
LAZDINS
from cercariae using two methods. The first method involves penetration of cercariae through rat skin and recovery of schistosomula 3-4 hr later as previously described (Stirewalt et al. 1966; Clegg and Smithers 1972). The second method originally reported by Ramalho-Pinto et al. (1974) involves mechanical transformation of cercariae to schistosomula induced by shearing in a Vortex mixer. After detachment by this procedure, cercarial bodies were separated from trails on Percoll gradients as outlined in Fig. 1. Five-day-old schistosomula were obtained by the following procedure. CBA mice (Jackson Laboratories, Bar Harbor, ME, USA) were injected intravenously (iv) with 2 x 10” mechanically transformed schistosomula or subcutaneously with 3 x lo3 cercariae. Five days later, the mice were sacrificed, the lungs removed and chopped, and schis[
FIG.
for the preparation schistosomula method of Ramalho-Pinto et al. (1974) with the newer technique employing bodies from tails by centrifugation on Percoll gradients. Schistosoma
1. Scheme
mansoni
of mechanical employing the as compared separation of discontinuous
ET AL.
tosomula recovered from lung fragments as described previously (Sher et al. 1974, 1978). In several experiments, lung schistosomula preparations were further purified by passage through nylon-wool columns followed by two cycles of sedimentation (20 min each) at Ig in 15ml conical centrifuge tubes (Butterworth et al. 1979). The purification of 5-day-old schistosomula using Percoll gradients is described under Results. Preparation of density gradients. Percoll (polyvinylpyrolidone-coated colloidal silica particles, starting density 1.135 g/ml) was purchased from Pharmacia Fine Chemicals (Piscataway, NJ, USA) (Pertoft et al. 1978). Isotonic solutions of Percoll of different densities were prepared by diluting the stock suspension with the appropriate volumes of 10x concentrated minimal essential medium (MEM) (Microbiological Associates, Walkersville, MD, USA) and distilled water. The solutions were then buffered with 1 M Hepes (Microbiological Associates) to a final concentration of 25 mM and the pH adjusted to 7.0-7.2 with 1 N NaOH. The densities (g/ml) calculated from manufacturers specifications were as follows: 1.044 (30%), 1.058 (40%), 1.068 (50%), 1.085 (60%), and 1.099 (70%). These solutions were stored at 4 C before use. To achieve sharp interfaces, 10 ml of each of these solutions was layered from the bottom of 50-ml plastic conical centrifuge tubes (Bioquest, BBL, Falcon Products, Becton-Dickinson, Cockeysville, MD, USA) using a lPgauge, 5-in.-long needle placed in the tube. This needle was connected to a lo-ml syringe via a three-way stopcock that allowed regulation of the flow rate of the Percoll solutions. The samples to be fractionated were layered at the top of the gradient in a volume of 5 ml of medium. The separation was achieved by low-speed centrifugation (35Og) for 10 min at 5 C. Five-milliliter samples were then collected from the bottom of the tube after perforation with a heated 16-gauge needle. The collected fractions were diluted in IO-vol of
Schistosoma
mansoni:
RAPID
MEM and the schistosomula pelleted by centrifugation. The samples were washed two additional times before further use. No attempt was made to determine the density of the collected fractions and they are designated according to the estimated percentage of Percoll which they contain. Assay of viability. The viability of schistosomula preparations were evaluated by the following criteria. (1) Direct microscopic examination of parasite motility and activity of flame cells. Parasites were scored as dead if they were immotile, nontranslucent, and had become granular and opaque in appearance. (2) Dye exclusion. Microscope slides were precoated with a drop of 0.1% toluidine blue solution in methanol, which was allowed to dry. A sample was placed on the slide and examined by light microscopy (Butterworth et al. 1979). Parasites were scored as dead when they were observed to take up large amounts of dye. (3) In vivo recovery. Precounted quantities of schistosomula (100-200) were injected iv into the tail veins of CBA/J mice. Six weeks later the animals were perfused (Smithers and Terry 1965) and the recovery of adult worms was determined.
ISOLATION
OF
SCHISTOSOMULA
41
were centrifuged in a discontinuous Percoll density gradient. A clear-cut separation of dead from live worms was observed (Fig. 2). The living organisms (which by morphology and dye exclusion were 100% viable) were found in the 50 and 60% interfaces. In contrast, the dead organisms were observed to band in the 30 and 40% interfaces. A similar separation was achieved when a mixture of live and killed (by exposure to 1 m&f N-maleimide) mechanically prepared schistosomula was applied to the gradient (data not shown). As can be seen in Fig. 2, the method fails to separate cercariae present in the preparation from living schistosomula. Separation of Mechanically Detached Cercarial Bodies from Tails Bodies and tails were prepared from lo5 cercariae by mechanical disruption, resuspended in 5 ml of MEM, and loaded on the top of individual gradients. After centrifugation, ten 5-ml fractions were collected and the number of bodies and tails in each fraction was enumerated. Results from a typical experiment are shown in Fig. 3. It can be seen that 90% of the cercarial bodies were found at the bottom of the tube in a
RESULTS
Purification of Schistosomula by Skin Penetration
Prepared
When Schistosoma mansoni cercariae are transformed to schistosomula by the skin penetration method, the preparations are usually contaminated with dead organisms (5-20%). This contamination becomes critical in experiments where the schistosomula are to be used as targets in in vitro killing experiments. Previously it was noticed that dead organisms are more difficult to pellet by centrifugation. This observation suggests that significant changes in density occur when schistosomula are killed. In order to test this hypothesis, preparations of skin-stage schistosomula highly contaminated with dead organisms
% PERCOU
2. Separation by centrifugation on Percoll of live from dead Schisrosoma mansoni schistosomula transformed from cercariae by skin penetration. A preparation of schistosomula consisting of 6% living schistosomula, 20% dead schistosomula, and 11% cercariae was loaded onto Percoll gradients. After centrifugation, 5-ml fractions were collected and the numbers of living and dead schistosomula (evaluated by morphologic examination and dye exclusion) and cercariae present in each fraction were counted. FIG.
42
LAZDINS
3i? ap
Cercariol
Bodies
Cercarial
Tails
50
25 0 30
40
50
60
70
% PERcoLL
FIG. 3. Separation of Schistosoma mansoni cercarial bodies from tails by centrifugation on Percoll. A population of freshly shed cercariae (105) was mechanically disrupted into bodies and tails and the preparation separated on Percoll as outlined in Fig. 1.
ET AL.
and 60% fractions and were highly contaminated with tails. This observation led us to examine the change in density undergone by cercarial bodies during their transformation to schistosomula. An example of an experiment demonstrating this density shift is shown in Fig. 4. It can be seen that after 150 min of incubation, the density distribution is very similar to that observed when schistosomula produced by skin penetration are separated on the gradient (Fig. 2). In addition, morphological examination (by Normarski interference microscopy) revealed that the majority of the larvae recovered from the gradient after incubation had lost their glycocalyx as well as the contents of their acetabular glands. No attempt was made to quantitate this relationship.
fraction contaminated with less than 1% tails. The few intact cercariae remaining after mechanical transformation were also found in this fraction. In contrast, most tails of j-Day-Old Schistosomula were found at the interface of the 40 and Purification from Contaminating Lung Tissue 50% Percoll fractions. The fraction conand Cells taining the cercarial bodies was washed twice in MEM and incubated for 2 hr at 37 Schistosomula were recovered from the C. Schistosomula prepared in this manner lungs of mice 5 days after intravenous inwere found to be 100% viable as judged by jection according to standard procedures morphologic examination and dye exclusion and were obtained in yields greater than 90% as compared with the yields of 30-40% obtained by means of the conventional procedure of Ramalho-Pinto et al. (1974). In later experiments it was found that the above protocol for the purification of mechanical schistosomula could be simplified by fractionating the sheared cercariae on single cushion of 70% Percoll (50,000 parasites/10 ml). After centrifugation (10 min 35Og), the cercarial tails were found at the top of the cushion and the bodies in a pellet at the bottom of the tube. The yields of schistosomula using this modified proce30 40 50 60 70 dure were 90% or greater and the purity % PERCOU excellent (better than 95%). FIG. 4. Density change in Schistosoma mansoni from cercarial bodies into If the suspension of tails and bodies was larvae during transformation mature schistosomula. Cercarial bodies (105) were infirst incubated to achieve the transformacubated in Earles/lactalbumin medium at 37 C and at tion to schistosomula, and then placed on the intervals indicated aliquots (2 x lo4 organisms) gradients, it was observed that the schis- were loaded onto individual Percoll gradients and the tosomula did not pellet to the bottom but mobility of the larvae in the gradients was analyzed instead were found in the interface of the 50 after centrifugation (10 min).
Schistosoma
t?lanSOtZi:
RAPID
(Sher et al. 1974). The worm preparations from 10 mice were resuspended in a total of 5 ml of MEM and placed at the top of the gradient, centrifuged, and processed. Most of the 5-day-old schistosomula (70%) were observed to band in the 50% Percoll fraction (Fig. 5). In contrast, the large fragments of lung tissue remained in the 30% fraction and most single cells were scattered in the 30 and 40% fractions. The viability of the lung stage schistosomula purified on Percoll gradients was found to be greater than 95% as determined by morphological examination and dye exclusion. This finding was corroborated by injecting mice with the isolated schistosomula and assaying the recovery of adult worms 6 weeks later. The results are presented in Table I. No significant differences in recovery were observed from animals that had received 5-day-old schistosomula prepared by the traditional method of nylon-wool liltration and from animals which received schistosomula purified on density gradients (Experiments 1 and 3) and indeed in one experiment (No. 2) the recovery from animals that had received 5-day-old schistosomula prepared by density gradient centrifugation was significantly higher than the recovery from animals that had received worms purified by the nylon-wool technique. DISCUSSION
Gradients of colloidal silica have been used successfully to isolate cells and sub-
ISOLATION
OF
SCHISTOSOMULA
30
40
50
60
70
% PERCOU
FIG. 5. Separation of S-day-old Schistosoma mansoni schistosomula from contaminating host tissue by centrifugation on Percoll. The inserts indicate the distribution of free lung cells or lung fragments (LF) on the gradient.
cellular particles of various types including liver cells, HeLa cells, viruses, lysozomes, mitochondria, etc. (Pertoft and Laurent 1977). Colloidal silica particles coated with polyvinylpyrolidone are nontoxic and offer advantages over conventional materials such as sucrose of metrizamide because of their low viscosity, low osmolality, and impermeability to biological membranes. In this paper we have described the use of this material to form discontinuous gradients which are employed to purify Schistosoma mansoni schistosomula of different developmental stages. Our data indicate that schistosomula isolated by centrifugation on Percoll exhibit no loss in viability, as judged by morphologic examination and dye exclusion and by their capacity to develop into adult worms after injection in mice (Table I). Furthermore, with most of the currently available methods for the iso-
TABLE I Infectivity of 5-Day-Old Schistosoma mansoni Schistosomula, Purified by Percoll Gradient Centrifugation Percent recovery of adult worms from mice injected with: Experiment 1 2 3
Percollpurified larvae
Nylon-wool purified larvae
P value
41 k 4.7 (5, 65)O 56 ?I 2.9 (7, 200) 25 k 1.7 (8, 170)
55 k 5.7 (5, 106) 41 ” 3.3 (7, 200) 25 k 1.3 (8, 170)
0.085 (NS)* 0.005 0.909 (NS)
a Results are presented as means k SEM. The numbers in parentheses refer respectively, to the number of mice perfused in each experiment and the average number of larvae injected per mouse. * Not statistically significant.
44
LAZDINS
lation and purification of schistosomula, yield is sacrificed for the sake of purity. With the use of the Percoll centrifugation technique, we have achieved high purity with yields often greater than 90%. The experiments reported here (Fig. 4) reveal that when cercariae are transformed in vitro to schistosomula, there is a simultaneous shift in the density of the cercarial bodies such that by 150 min of incubation the density of the organisms is indistinguishable from that of skin-stage schistosomula. Acetabular gland secretion, the loss of the cercarial glycocalyx, and changes in membrane permeability are known to occur during the same period (Stirewalt and Uy 1969; Gazzinelli et al. 1973) and probably underlie the density transition observed. For this reason, parasite density, as determined by Percoll centrifugation, may prove to be a useful analytical parameter of irt vitro transformation. A final application of the Percoll separation technique described in the present study was the use of the method to purify schistosomula from contaminating host tissue. Previous attempts to isolate 5-day-old parasites free of cells and tissue fragments by means of sedimentation and filtration through nylon-wool columns (Butterworth et al. 1979) have met with only partial success. By means of density centrifugation on Percoll, it is now possible to isolate 5-dayold schistosomula in sufficient purity and yield to allow biochemical analysis. For instance, it should now be feasible to chemically characterize the parasite antigens as well as host molecules associated with the larval surface, a problem which until now has been impractical to pursue because of the contamination of schistosomulum preparations with host tissue. ACKNOWLEDGMENTS This work was supported by grants from the Rockefeller Foundation, The Clark Foundation, and the U.S. National Institute of Allergy and Infectious Diseases. We thank John Samuelson for his helpful discussions and suggestions and Christine Sleiman for her assistance in the preparation of this manuscript.
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