EXPERIMENTAL PARASITOLOGY 48,421-431 (1979)
Babesia rodhaini, Babesia bovis, and Babesia bigemina: Analysis and Sorting of Red Cells from Infected Mouse or Calf Blood by Flow Fluorimetry Using 33258 Hoechst J. HOWARD*,' AND BARRY J. RODWELL?
RUSSELL
*Laboratory of Immunoparasitology, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital P.O., Melbourne 3050, and tTick Fever Research Center (of the Animal Research Institute, Yeerongpilly) Wacol, Brisbane, Queensland 4076, Australia
(Accepted for publication 18 July 1979) HOWARD, R. J., AND RODWELL, B. J. 1979. Babesia rodhaini, Babesia bovis, and Babesia bigemina: Analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst. Experimental Parasitology 48,421-431. The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent
bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments. INDEX DESCRIPTORS: Babesia rodhaini; Babesia bovis; Babesia bigemina; Protozoa, parasitic; Babesiosis; BALB/c mice; Bos taurus calves; Cell separation; Erythrocytes; Fluorescent cell sorting; DNA content.
INTRODUCTION
Babesia spp. infections in the vertebrate host are characterised by anemia, hemoglobinemia, and hemoglobinuria, and in many cases death of the host. These symptoms are related to the multiplication of the parasite within host red blood cells, and repeated cycles of red cell invasion, intraerythrocytic growth, and multiplication followed by red cell lysis and reinvasion (Mahoney 1972, 1977). The number of daughter parasites produced per infected cell is generally two, or occasionally four. ’ Correspondence address: Malaria Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Md. 20014.
Multiple invasion of red cells in severe infections may produce pluriparasitized cells which resemble schizont-infected cells typical of malaria infections (Ewing 1965). In order to study the detailed biochemistry and immunogenicity of the different forms of cells infected with Babesia spp., techniques for the physical separation of these cells are required. Methods which rely on possible differences in buoyant density or sedimentation velocity of infected and uninfected cells have failed with the bovine (Mahoney 1972) and murine parasites (R. J. Howard, unpublished results), although some separation can be achieved with Babe& cunis and cells infected with B. cubulli (Watkins 1962; Hirsh et al. 1969).
421 0014-4894/79/060421-l1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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With B. bovis infections, but not with B. bigemina, it is possible to purify infected cells by exploiting their greater resistance to hypotonic lysis than infected cells (Mahoney 1967). Unfortunately this procedure may modify the surface of the purified infected cells. We have recently described a cell sorting technique for the separation of red cells infected with the rodent malaria parasite Plasmodium berghei based on their DNA content (Howard and Battye, in press; Howard et al. 1979). This technique employs the DNA binding dye 33258 Hoechst which binds specifically to A-T-rich DNA sequences within living or fixed cells with a large increase in fluorescence yield (Latt and Wohlleb 1975). This property of 33258 Hoechst was used to identify Babesia canis by fluorescence microscopy of blood smears (Lammler and Schutze 1969), and more recently has been applied in estimating parasitemia with Babesia spp. and Anaplasma spp. in cattle blood (B. J. Rodwell and R. J. Howard, in preparation). It is possible with the cell sorting technique to separate 100% pure uninfected cells (which are nonfluorescent) and different stages of cells infected with Plasmodium spp. that contain different multiples of the DNA content of a singly infected cell. The sorted infected cells retain the capacity to infect mice. We have now applied the technique to the separation of parasitized red blood cells from several Babesia infections: B. rodhaini infections of BALB/c mice, and B. bovis and B. bigemina infections of Bos taurus calves. We report that provided the optimal dye concentration for DNA labeling has been established, separation of red cells infected with Babesia spp. can also be achieved. MATERIALS
AND METHODS
Host animals. Male or female BALB/c mice, 6-8 weeks old, were derived from specific pathogen-free conditions and maintained at The Walter and Eliza Hall In-
stitute, Melbourne, as described elsewhere (Mitchell et al. 1976). The mice were infected with Babesia rodhaini by inoculation with heavily infected blood from infected donor animals (Cox et al. 1977). Bos taut-us calves, 5-6 weeks old, were maintained under tick-free conditions at The Tick Fever Research Center near Brisbane, and after splenectomy were infected with Babesia bovis or Babesia bigemina by standard methods (Callow and Tammemagi 1967). The calves were free of all other blood infections, as judged by examination of thick or thin smears by light microscopy. Parasitized blood was collected into Alsever’s solution from the retro orbital plexus of mice, and from the jugular vein or carotid artery of calves (Callow and Mellors 1966). Mouse blood was washed and labeled with the DNA dye immediately after bleeding, whereas infected bovine blood was packed in ice and air freighted from Brisbane to Melbourne where it was washed twice at 4 C in phosphate buffered saline (PBS: bovine tonicity, 20 m&f sodium phosphate, 0.125 M NaCl, pH 7.3; mouse tonicity, 20 mM sodium phosphate, 0.149 M NaCl pH 7.3), then stored overnight at 4 C in PBS with 1 mg/ml added D(-)-glucose. The bovine blood was then rewashed twice before DNA labeling and cell sorting. The total time between bleeding the calves and DNA labeling never exceeded 25 hr. Preliminary experiments established that storage of mouse blood infected with B. rodhaini under the same conditions for 24 hr caused less than 5% loss of cells from the fluorescent cell peaks. Labeling cells with 33258 Hoechst. The 33258 Hoechst was obtained from Riedel-de Hadn AG, Seelze-Hannover and was used as described previously (Howard et al. 1979). Red blood cells were washed three or four times in mouse or bovine tonicity PBS as appropriate and the buffy coat was removed by aspiration. In some experiments the leukocyte contamination of mouse blood was reduced further (to less than
Babesia
spp.:SORTING OF INFECTED
0.05% of the total cells) by passage of the cells through a sulfoethyl cellulose column (Howard& al., 1978), although depletion of leukocytes was subsequently shown to be unnecessary. Washed red cells, 6 x 107, were resuspended in Eisen’s balanced salt solution [EBSS: Tris 5 m&f, sodium phosphate 5 n&f, MgSO, 1 mM, CaCl, 2 mM, KC1 6 mA4, with either 0.15 M NaCl (mouse tonicity) or 0.125 M NaCl (bovine tonicity)], plus 1 mg/ml D( -)-glucose, followed by addition of a sample of 100 or 200 pM 33258 Hoechst in EBSS plus glucose to the desired final dye concentration in 10ml total volume. Samples were immediately placed in darkness and incubated at 37 C in a humidified atmosphere of 10% CO, in air for 2 hr. At 37 C the dye enters living cells and binds reversibly to nuclear DNA (Arndt-Jovin and Jovin, 1977). The cells were then rapidly washed three times in PBS at 4 C in darkness, resuspended finally in 10ml, and sorted at this concentration. Cells were stored and sorted at 4 C in darkness. Cell sorting and analysis. The details of operation of the Becton Dickinson FACS II fluorescence activated cell sorter (FACS) and method of separation of fluorescent light from scattered laser light by optical filters have been described elsewhere (Howard and Battye, in press). Cells were sorted on the basis of their integrated low angle light scatter (approx. 1- 15”) and characteristic 33258 Hoechst fluorescence (350-nm excitation maximum and 460-nm emission maximum). The values of both parameters were expressed in 256 channel memory segments of the FACS II. The sensitivity of cell fluorescence detection could be increased by increasing the photomultiplier voltage or pulse amplifier gain setting. The sorted stream was directed into a siliconized glass tube containing 1 to 2 ml of PBS with 10% (v/v) fetal calf serum. Cells were collected by centrifugation (300g x 7 min, 4 C) and resuspended in 100 ~1 of the same medium with a fine bore Pasteur
RED CELLS
423
pipet. A drop of the suspension (lo-50 ~1) was applied to a glass slide, air dried, and stained with Giemsa’s stain after fixation in 90% (v/v) methanol. Infectivity studies. The effect of 33258 Hoechst labeling and sorting on the infectivity of blood cells with B. rodhaini was tested by the following qualitative method. Between 3 x 10” and 9 x 10” cells were sorted from the center of each of the fluorescent peaks Pl, P2, and P3 (Table II) and also from the nonfluorescent peak, diluted in PBS and lo* cells injected directly (iv) into BALB/c mice. As the steps prior to injection were performed on ice, the dye would have remained bound to parasite DNA (Howard et al. 1979). Several days after injection smears of peripheral blood from these mice were Giemsa stained and examined by light microscopy in order to verify the infectivity of injected parasites. RESULTS
Cell Sorting on the Basis of Low Angle Light Scatter The low angle scatter distributions of red cells from normal mouse or calf blood occasionally differed from the samples infected with Babesia spp. However, the differences were not reproducible, and generally too small to be exploited as a parameter for the sorting of different red blood cell populations . The low angle scatter distribution of B. rodhaini-infected blood depleted of leukocytes by repeated washing and aspiration of the buffy coat is compared in Fig. 1 with the scatter distribution of blood taken into Alsever’s solution and sorted directly without any attempt to remove leukocytes. Buffy coat removal effectively removed the leukocytes which are characterized by a higher low angle scatter intensity than red cells. In order to restrict fluorescence analysis to red blood cells and exclude smaller particles such as free parasites and parasite nu-
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species. In general, at the lowest dye concentrations tested the proportion of total cells which was fluorescent was much less than the degree of parasitemia, and, as the concentration was raised the proportion of fluorescent cells increased and the intensity of each fluorescent cell peak also increased. By using 100 pM dye for B. rodblood the haini and B. bovis-infected number of fluorescent cells was maximal, the fluorescence intensity of each of the fluorescent cell peaks was maximal and the resolution of the different peaks was also 0 optimal (Figs. 2A and C). The fluorescent 0 64 I20 192 256 profiles for B. bigemina-infected blood difRELATIVE LOU ANGLE SCATTER FIG. 1. The effect of leukocyte depletion on the low fered in that two fluorescent cell peaks angle scatter distribution of mouse blood infected with were clearly resolved at l-10 pM dye but Babesia rodhaini. Infected blood of 48% parasitemia at higher dye concentrations the two peaks was either depleted ofleukocytes by five washes in PBS merged into a broad peak centered at a and aspiration of the buffy coat (A), or, mixed directly with Alsever’s solution (B) prior to the analysis of low fluorescence intensity slightly less than the angle scatter. most fluorescent peak (Fig. 2B). The number of fluorescent cells was identical clei, upper and lower limits were set to the over the range of dye concentration tested for B. bigemina. A dye concentration of 2 intensity of low angle scattered light. pM was chosen for subsequent experiDual Parameter Cell Sorting ments with B. bigemina in order to resolve The effects of different dye concentra- the maximum number of different cell tions on the fluorescent cell distribution are types. Normal calf or mouse blood which had summarized in Fig. 2 for each of the Babesia
4
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0 64 128 192 256 RELATIVE FLUORESCENCE
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a 0 64 I28 192 256 RELATIVE FLUOAESCEMCE
FIG. 2. Comparison of the fluorescent cell profiles for bloods infected with Babesia rodhaini, Babesia bigemina, and Babesia bovis after labeling with different concentrations of 33258 Hoechst (PM dye concentrations indicated in figure). (A) B. rodhaini-infected mouse blood (84% parasitemia) labeled with 0.1 - 100 @4 dye. (B) B. bigemina-infected calf blood (2.5% parasitemia) labeled with 2-100 FM dye. (C) E. bovis-infected calf blood (5.4% parasitemia) labeled with 2-100 pM dye.
Babesia
spp.: SORTING OF INFECTED
RED CELLS
425
TABLE I Comparison of the Proportion of Fluorescent Cells Labeled by 33258 Hoechst with the Parasitemia Determined by Light Microscopy
Parasite Babesia rodhaini
Sample 1
2 Babesia bovis
1
Babesia bigemina
2 1 2
Percentage fluorescent cells”
Percentage of cells parasitized (light microscopy)”
55.3 12.2 4.9 0.6’ 5.5 0.13’
49 10 4 channel 11 for B. rodhaini and > channel 25 for B. bovis and B. bigemina.
Ir Thin smears of infected blood were Giemsa stained and 1000 cells examined for parasites in several fields. ’ The fluorescent cell distributions for these samples of very low parasitemia were identical to those of higher parasitemia.
been leukocyte depleted was incubated at the dye concentrations chosen for optimal labeling of infected blood. Less than 0.1% of the total cells were fluorescent. Infected blood which had not been treated with the dye was also nonfluorescent (~0.1% fluorescent cells). The percentage of parasitized cells determined by light microscopy was almost identical to the percentage of fluorescent cells under optimal labeling conditions (Table I). The consistent finding that the percentage parasitemia determined by light microscopy was less than the percentage of fluorescent cells (Table I) may have been due to difficulty in identifying small stained Babesia parasites. Cells were sorted from the center of each peak in the fluorescent cell distribution and examined by light microscopy after Giemsa staining. The fluorescence properties of each of the selected populations are shown in Fig. 3 for each Babesia infection. Table II summarizes the light microscopy analysis of these populations. B. r&z&i-infected blood usually exhibited four fluorescent cell peaks in addition to the nonfluorescent cells (Fig. 3A). The fluorescent peaks are denoted Pl, P2, and P3 in Table II in order of increasing fluorescence intensity. The ratio of the fluores-
cence intensities of the maxima of each of the peaks Pl to P3 was 1.0:2.0:3.0 (Fig. 3A). As saturation levels of dye bound to DNA had been reached (Fig. 2A) this ratio
0 64 256 I211 192 RELATIVE FLUOflESCLWCE
FIG. 3. Selection of fluorescent cell peaks for sorting after optimal conditions of cell labeling. (A) Babesia rodhaini-infected mouse blood (79% parasitemia) labeled with 100 KM dye. With photomultiplier voltage (PV) of 575 and gain of 0.5, the fluorescent channels selected for sorting Pl, P2, and P3 were, respectively, 29-51.67-93, and 108-137, as indicated. (B) Babesia bovis-infected calf blood (4.3% parasitemia) labeled with 100FM dye. The PV was 600 and gain 0.5. The fluorescent channels selected for sorting Pl (32-85) are indicated. (C) Babesia bigemina-infected calf blood (1.2% parasitemia) labeled with 2 pM dye. The fluorescent channels selected for sorting Pl (56-96) and P2 (112-184) are shown for sorting under the same conditions as B.
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TABLE II Analysis of Red Cells from Babesia-Infected Blood Sorted on the Basis of DNA Content” Percentage of sorted cells
Parasite Babrsia
rodhaini
Babesia bovis
Babesia bigemina
Number of parasite nuclei per red cell
Sorted cells (channel limits) Nonfluorescent Fluorescent Pl” P21’ P3” Nonfluorescent Fluorescent Pl Nonfluorescent Fluorescent Pl P2
0
1
2
(O-3)
100
0
0
0
(29-51) (67-93) (108- 137) (O-5)
5 1 0 100
92 26 5 0
3 60 9 0
0 13 86 0
(32-85) (O-5)
1 100
94 0
5 0
0 0
2 0
96 81
2 19
0 0
(56-96) (112-184)
>3
” Cells from infected blood labeled under the same conditions as in Fig. 3. Cells, lo’- lo’, were sorted from the center of each peak (between the designated channel limits), methanol fixed on glass slides and examined by light microscopy after Giemsa staining. Six hundred cells were analyzed for each sample. ’ Smears of each of these sorted fluorescent cell populations indicated a significant proportion of parasites to be extracellular (2- 15% of the parasites in the sorted population in different experiments).
suggested that the DNA content of infected cells increased by integral amounts, i.e., integral numbers of parasites. No infected cells were seen in the nonfluorescent fraction. PI of B. rodhaini-infected blood consisted predominantly of red cells with a single parasite, whereas 60% of cells in P2 contained two parasite nuclei. The majority of the binucleate cells in P2 (>90%) contained two separate parasites. The cells with a3 nuclei in P3 contained morphologically diverse parasites, but approximately 80% of these infected cells contained 3 parasites each with one nucleus. Both P2 and P3 contained multinucleate parasites, but these occurred infrequently. The maximum number of parasites seen in a single cell was 8. Samples of B. rodhainiinfected blood of low parasitemia (~10%) contained a much lower proportion of fluorescent cells in the highly fluorescent peaks (aP3). B. bovis-infected blood (Fig. 3B) had a relatively simple distribution of fluorescent cells compared with B. rodhaini (Fig. 3A) exhibiting a single broad fluorescent peak
(Pl in Table II). This peak consisted of >90% singly infected cells with a small proportion of cells containing two parasites (Table II). After labeling B. bigemina-infected blood with 2 ,uM dye we sorted and examined the separate peaks (Fig. 3C). The peak of lowest fluorescence intensity (Pl in Table II) contained >95% singly infected red cells. The second peak (P2) was also predominantly singly infected cells, but in addition contained a significant proportion of cells with two parasites (5-20% in different samples (Table II)). The singly infected cells in PI and P2 were morphologically indistinguishable by light microscopy. The nonfluorescent cells consisted exclusively of uninfected red cells in both of the bovine infections (Table II). Infectivity
of Sorted Cells from
Babesia
rodhaini Infection Following injection of IO’ cells from Pl, P2, or P3 (4 mice per group), all animals died of B. rodhaini infection after IO-13 days. Injection of IO’ cells from the
Eobesia spp.: SORTING OF INFECTED
nonfluorescent peak also resulted in lethal B. rodhuini infection in a proportion of mice (death at 13- 14 days of l/4 and 214mice in two experiments). By examination of the fluorescence distribution of B. rodhuini-infected blood at very high sensitivity (Fig. 4) we could identify a minor population of cells with very low fluorescence which overlapped the nonfluorescent cell peak. Light microscopy analysis of cells sorted from the low fluorescence peak indicated that all of these cells were red cells which stained blue after Giemsa staining (and may therefore be reticulocytes), whereas cells sorted from channels O-15 of the nonfluorescent peak consisted of erythrocytes. Furthermore, approximately 5% of the cells in the low fluorescence peak contained a single very small parasite (red nucleus and very small ring of blue cytoplasm). Parasites were not detected in the sorted nonfluorescent cells. Injection of lo* cells sorted from the low fluorescence peak (Fig. 4) resulted in Babesia rodhaini infection and death at 14- 16 days of 3/4 mice in two experiments. However, infective particles were still 4r
:.
L 0
I
64
I
1
128 192 RELATIVE FLUORESCENCE
I 256
FIG. 4. Identitication of a population of Babesia rodhaini-infected cells which are poorly resolved from uninfected cells after 33258 Hoechst labeling. Infected blood of 53% parasitemia was labeled with 1OOpMdye and the fluorescent cell distribution examined with a PV of 625 and gain of 1.0. Under these conditions of increased sensitivity, nonfluorescent cells were sorted from channels 0- 15 and a population of cells with very low fluorescence was sorted from channels 36-62. Cells containing one parasite nucleus, comparable to the peak of lowest fluorescence intensity in Fig. 3A, are shown between channels 192and 256 in this figure.
RED CELLS
427
present in the nonfluorescent peak, as injection of lo* cells sorted from channels 0- 15 (Fig. 4) also produced infection in 2/4 mice (two experiments). These mice did not die until 18-20 days after injection. DISCUSSION
Cells infected with Babesiu spp. can be separated from uninfected cells and from each other on the basis of their DNA content after staining with 33258 Hoechst, thereby allowing a new method for the physical separation of red blood cells from infected blood. The essential features of this method are as follows: (1) preliminary purification of red cells by leukocyte depletion and washing (Fig. l), (2) incubation of red blood cells with the dye at 37 C. The concentration of dye used is critical in determining the final proportion of fluorescent cells and fluorescent cell profile (Howard et al. 1979; and Fig. 2). Provided the cells are maintained on ice and in darkness the dye remains bound to DNA for at least several hours (Arndt-Jovin and Jovin 1977; Howard et al. 1979); (3) cell sorting on the FACS using both low angle scatter and fluorescence parameters to restrict analysis to red cells. The dye can be removed from sorted cells by incubation at 37 C (Arndt-Jovin and Jovin, 1977). Some parasite viability is retained even when the dye is not removed before inoculation of the host. The concentration of 33258 Hoechst required for maximal fluorescence intensity for each peak in the distribution was 100 pM for each of the three Bubesiu species (Fig. 2), although this concentration was not chosen for separation of the cell types in B. bigeminu-infected blood. Two cell populations in B. bigeminu-infected blood were resolved at lower dye concentrations (2-10 pM) but merged at the higher saturating dye concentration. In contrast, maximal fluorescence intensity for each fluorescent cell peak in P. berghei-infected blood was achieved with 2 ~(LM dye in the
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same incubation medium (Howard et al. 1979). The morphology of Babesia varies, and there is debate as to the exact developmental sequence in the blood (Riek 1964, 1966; Holbrook et al. 1968; Kreier et al. 1975; Potgieter, 1977; Rudzinska and Trager, 1977). Light and electron microscope studies by these workers suggest explanations for the different populations of infected cells sorted on the basis of DNA content in the present work. B. rodhaini-infected cells were sorted into at least three fluorescent cell peaks (Fig. 3A). The fluorescence intensity of the center of each of these fluorescent peaks increased in an exactly integral ratio. Examination of the cells in Pl, P2, and P3 (Table II) by light microscopy confirmed that the majority of infected cells in these peaks contained, in order 1, 2, or 3 parasites. It is possible that a proportion of cells with fluorescence aP3 contained parasites derived from repeated merozoite invasion of a single red cell. The extremely high parasitemia of the B. rodhaini samples examined (30-80%) may have led to multiple invasion. Cytofluorimetric analysis of the DNA content of Plasmodium berghei merozoites and intracellular parasites (Bahr and Mikel, 1972), suggested that the DNA content of the merozoite was half that of young trophozoite forms. Although it could be concluded on this basis that the parasite DNA is very rapidly replicated after merozoite entry, the limits of sensitivity of the technique used plus the small amount of parasite DNA preclude a definitive conclusion. Biosynthetic labeling studies by Gutteridge and Trigg (1971) indicated that DNA synthesis in Plasmodium spp. occurs during the immature and mature trophozoite stages prior to schizogony and therefore precedes cytoplasmic and nuclear division. We observed a small number of B. rodhainiinfected cells with up to eight parasite nuclei in the third fluorescent peak (P3). The
RODWELL
DNA content of the merozoite (i.e., the parasite in mature heavily infected cells) may therefore be less than that of singly infected cells in PI. Infected cells were also identified in P2 with only a single parasite nucleus yet twice the fluorescence of uninucleated cells in Pl (Table II). These results suggest that the DNA content of Babesiainfected cells may also increase without cytoplasmic division. Other methods will be required to establish the exact time sequence of DNA synthesis between merozoite invasion and nuclear division. It is possible that the small parasite forms seen in some of the very weakly fluorescent reticulocytes (Fig. 4) represent recently invaded merozoites. As these parasiteinfected cells were no more fluorescent than the reticulocytes, their DNA would appear to be refractory to 33258 Hoechst labeling under the usual labeling conditions. At least some of these parasite forms must be viable, as inoculation of cells from the weakly fluorescent peak, and the nonfluorescent cells which overlap this peak infected mice. Sorting on the basis of low angle light scatter excluded the possibility that the viable particles in this fraction were merozoites. This result was unexpected in view of the apparent absence of parasites as judged by light microscopy of the nonfluorescent cells (Table II). Our experiments with P. berghei-infected blood sorted after 33258 Hoechst staining (Howard and Battye, in press; Howard et al. 1979) demonstrated that even when 4 x lo5 nonfluorescent cells were injected per mouse, P. berghei infection did not result. Fluorescent cells did, on the other hand, cause lethal P. berghei infection upon injection. With 100 pM 33258 Hoechst, B. bigemina-infected blood and B. bovisinfected blood each contained only one major fluorescent cell population which consisted predominantly of singly infected cells (Table II). The presence of a small number of cells containing two parasites in
Babesia spp.: SORTING
OF INFECTED
this fluorescent peak indicates that the DNA content of the singly infected cells was equivalent to at least two merozoites. These parasites are thought to multiply by a budding process to produce one pair of pyriform parasites which leave the cell to reinvade other red cells (Riek, 1966). The cells containing two parasites in this fluorescent peak would therefore be mature infected cells about to rupture. The budding process of Babesia differs from schizogony typical of Plasmodium in that nuclear division is completed at a very late stage of merozoite development, with little if any nuclear material remaining in the remnant of the mother cell (Potgieter, 1977). By contrast, after completion of schizogony in Plasmodium spp., considerable nuclear material remains in the residual parasite body (Aikawa, 1966). Infected cells containing parasites with less DNA than the major fluorescent peak (i.e., equivalent to a merozoite) were not seen. This suggests that rapid doubling of the parasite DNA content after red cell invasion may be a common feature of Babesia and Plasmodium infections. Another common feature might be the delay between DNA replication and nuclear and cytoplasmic division of the parasite. Very few red blood cells from B. bovis or B. bigemina-infected blood had fluorescence greater than the major fluorescent peak. Although these highly fluorescent cells were not examined by light microscopy, they may have contained three or four parasites as a result of multiple infection. One unusual feature of the fluorescent cell profile for B. bigemina-infected blood was the resolution of two infected cell populations at dye concentrations less than the saturation level (Fig. 2B). Light microscopy analysis of the two populations failed to indicate significant differences in gross morphology of these parasites after Giemsa staining. However, the greater prevalence of cells with two parasites in the more highly fluorescent peak was a consistent
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CELLS
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difference (Table II). These two cell populations appear to have different “affinities” for 33258 Hoechst such that after incubation in 2 pM dye for 2 hr they bound different amounts of dye. Incubation in 100 pM dye for the same period resulted in saturation binding for both populations. The slight decrease in fluorescence intensity of the P2 cells seen after treatment with 100 pM dye, compared to their fluorescence intensity after 2 pM dye (Fig. 2B), may be similar to the drop in fluorescence intensity of P. berghei-infected cells at supraoptimal dye concentrations (Howard et al. 1979) a phenomenon also observed when isolated DNA is labeled with high concentrations of the dye (Latt and Stetten 1976). The cell sorting method yields 99-100% pure uninfected cells from Babes&infected blood and 90% pure infected cells. Approximately 1.5 x lo6 uninfected cells, and 3-5 x lo” infected cells from the center of each fluorescent peak, can be sorted per hour with a 50% parasitemia of B. rodhaini. The number of infected cells collected per hour is proportionally fewer for B. bovis and B. bigemina-infected samples in which parasitemias reach a maximum of lo- 15%. These cell numbers are sufficient for microbiochemical analyses. However the method cannot resolve all of the morphological stages seen in infected blood samples. With B. bigemina-infected blood, at least 15 morphological forms of infected cells were described by light microscopy (Riek 1964). Separation on the basis of DNA content fails to resolve some cells of identical DNA content with obviously different morphologies-for example, uninucleate trophozoite-infected cells and cells containing two merozoites (Table II). Further advances in flow systems technology such as sorting on the basis of DNA content and a parameter such as high angle light scatter which is related to complex aspects of internal cell architecture, may resolve additional subpopulations of infected cells. The present method is being used to
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separate uninfected and infected cells from bovis-infected blood after radioiodination of cell surface proteins to enable pathological and immunological studies.
Babesia
ACKNOWLEDGMENTS
erinary Research
This work was supported by the UNDPiWorld Bank/WHO Special Program for Research and Training in Tropical Diseases and by the Australian National Health and Medical Research Council. The expert assistance of Dr. F. Battye in operating the FACS is gratefully acknowledged. The advice of Drs. L. L. Callow and G. F. Mitchell in preparing the manuscript is greatly appreciated. We are also grateful to Joan Curtis for technical assistance.
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of Protozoology
HIRSH, D. C., HICKMAN, R. L., BURKHOLDER,C. R.,
29, 297-303.
HOWARD, R. J., AND BATTYE, F. L. Plasmodium berghei-infected red cells sorted according to DNA content. Parasitology 66, in press. HOWARD, R. J., BATTYE, F. L., AND MITCHELL, G. F. 1979. Plasmodium-infected blood cells analysed and sorted by flow fluorimetry using the deoxyribonucleic acid binding dye 33258 Hoechst. Journal
of Histochemistry
and Cytochemistry
27,
803-813. HOWARD, R. J., SMITH, P. M., AND MITCHELL, G. F. 1978. Removal of leucocytes from red cells in Plasmodium berghei-infected mouse blood and puriiication of schizont-infected cells. Annals of Tropical Medicine
and Parasitology
72, 573 -575.
KREIER, J. P., GRAVELY, S. M., SEED, T. M., SMUCKER,R., AND PFISTER, R. M. 1975. Babesia sp.: The relationships of stages of development to structure of intra- and extracellular parasites. Tropenmedizin und Parasitologie 26, 9- 18. LAMMLER, G., AND SCHUTZE, H. R. 1969. Vitalfluorchromierung tierscher zellkerne mit einem neuen fluorochrom. Naturwissenschaften 56, 286. LATT, S. A., AND STETTEN, G. 1976. Spectral studies on 33258Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. Journal of Histochemistry and Cytochemistry
24, 24-33.
LATT, S. A., AND WOHLLEB, J. C. 1975. Optical studies on the interaction of 33258 Hoechst with DNA, chromatin and metaphase chromosomes. Chromosoma 52, 297-316. MAHONEY, D. F. 1967. Bovine babesiosis: The passive immunization of calves against Babesia argentina with special reference to the role of complement Parasitology 20, fixing antibodies. Experimental 232-241. MAHONEY, D. F. 1972. Immune response to hemoprotozoa. II. Babesia spp. In “Immunity to Animal Parasites” (E. J. Soulsby, ed.), pp. 301-341. Academic Press, New York/London. MAHONEY, D. F. 1977. Babesia of domestic animals. In “Parasitic Protozoa,” Vol. IV, pp. l-52. Academic Press, New York/London. MITCHELL, G. F., HOGARTH-SCOTT,R. S., EDWARDS, R. D., LEWERS,H. M., COUSINS,G., AND MOORE, T. 1976. Studies on immune responses to parasite antigens in mice. I. Ascaris suum larvae numbers and anti-phosphorylcholine responses in mice of
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spp.: SORTING OF INFECTED
various strains and in hypothymic mice. Infernational Archives
of Allergy
and Applied Immunology
52, 64-n. POTGIETER, F. T. 1977. “The Life Cycle of Babesia bovis and Babesia bigemina in Ticks and Cattle in
South Africa.” Ph.D. Thesis. Rand Afrikaans University. RIEK, R. F. 1964. The life cycle of Babesia bigemina (Smith & Kilborne, 1893) in the tick vector Boophilus microplus (Canestrini). Australian Journal of Agricultural Research 15, 802-821. RIEK, R. F. 1966. The life cycle of Babesia argentina
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(Lingnieres, 1903) (Sporozoa: Piroplasmidea) in the tick vector Boophilus microplus (Canestrini). Australian
Journal
of
Agricultural
Research
17,
247-254. RUDZINSKA, M. A., AND TRACER, W. 1977. Formation of merozoites in intraerythrocytic Babesia microti: An ultrastructural study. Canadian Journal of Zoology 55, 928-938. WATKINS, R. G. 1962. A concentration and staining technique for diagnosing equine piroplasmosis. Journal of the American Veterinary sociation 141, 1330-1332.
Medicine
As-
Babesia rodhaini, Babesia bovis, and Babesia bigemina: Analysis and Sorting of Red Cells from Infected Mouse or Calf Blood by Flow Fluorimetry Using 33258 Hoechst J. HOWARD*,' AND BARRY J. RODWELL?
RUSSELL
*Laboratory of Immunoparasitology, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital P.O., Melbourne 3050, and tTick Fever Research Center (of the Animal Research Institute, Yeerongpilly) Wacol, Brisbane, Queensland 4076, Australia
(Accepted for publication 18 July 1979) HOWARD, R. J., AND RODWELL, B. J. 1979. Babesia rodhaini, Babesia bovis, and Babesia bigemina: Analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst. Experimental Parasitology 48,421-431. The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent
bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments. INDEX DESCRIPTORS: Babesia rodhaini; Babesia bovis; Babesia bigemina; Protozoa, parasitic; Babesiosis; BALB/c mice; Bos taurus calves; Cell separation; Erythrocytes; Fluorescent cell sorting; DNA content.
INTRODUCTION
Babesia spp. infections in the vertebrate host are characterised by anemia, hemoglobinemia, and hemoglobinuria, and in many cases death of the host. These symptoms are related to the multiplication of the parasite within host red blood cells, and repeated cycles of red cell invasion, intraerythrocytic growth, and multiplication followed by red cell lysis and reinvasion (Mahoney 1972, 1977). The number of daughter parasites produced per infected cell is generally two, or occasionally four. ’ Correspondence address: Malaria Section, Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Md. 20014.
Multiple invasion of red cells in severe infections may produce pluriparasitized cells which resemble schizont-infected cells typical of malaria infections (Ewing 1965). In order to study the detailed biochemistry and immunogenicity of the different forms of cells infected with Babesia spp., techniques for the physical separation of these cells are required. Methods which rely on possible differences in buoyant density or sedimentation velocity of infected and uninfected cells have failed with the bovine (Mahoney 1972) and murine parasites (R. J. Howard, unpublished results), although some separation can be achieved with Babe& cunis and cells infected with B. cubulli (Watkins 1962; Hirsh et al. 1969).
421 0014-4894/79/060421-l1$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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With B. bovis infections, but not with B. bigemina, it is possible to purify infected cells by exploiting their greater resistance to hypotonic lysis than infected cells (Mahoney 1967). Unfortunately this procedure may modify the surface of the purified infected cells. We have recently described a cell sorting technique for the separation of red cells infected with the rodent malaria parasite Plasmodium berghei based on their DNA content (Howard and Battye, in press; Howard et al. 1979). This technique employs the DNA binding dye 33258 Hoechst which binds specifically to A-T-rich DNA sequences within living or fixed cells with a large increase in fluorescence yield (Latt and Wohlleb 1975). This property of 33258 Hoechst was used to identify Babesia canis by fluorescence microscopy of blood smears (Lammler and Schutze 1969), and more recently has been applied in estimating parasitemia with Babesia spp. and Anaplasma spp. in cattle blood (B. J. Rodwell and R. J. Howard, in preparation). It is possible with the cell sorting technique to separate 100% pure uninfected cells (which are nonfluorescent) and different stages of cells infected with Plasmodium spp. that contain different multiples of the DNA content of a singly infected cell. The sorted infected cells retain the capacity to infect mice. We have now applied the technique to the separation of parasitized red blood cells from several Babesia infections: B. rodhaini infections of BALB/c mice, and B. bovis and B. bigemina infections of Bos taurus calves. We report that provided the optimal dye concentration for DNA labeling has been established, separation of red cells infected with Babesia spp. can also be achieved. MATERIALS
AND METHODS
Host animals. Male or female BALB/c mice, 6-8 weeks old, were derived from specific pathogen-free conditions and maintained at The Walter and Eliza Hall In-
stitute, Melbourne, as described elsewhere (Mitchell et al. 1976). The mice were infected with Babesia rodhaini by inoculation with heavily infected blood from infected donor animals (Cox et al. 1977). Bos taut-us calves, 5-6 weeks old, were maintained under tick-free conditions at The Tick Fever Research Center near Brisbane, and after splenectomy were infected with Babesia bovis or Babesia bigemina by standard methods (Callow and Tammemagi 1967). The calves were free of all other blood infections, as judged by examination of thick or thin smears by light microscopy. Parasitized blood was collected into Alsever’s solution from the retro orbital plexus of mice, and from the jugular vein or carotid artery of calves (Callow and Mellors 1966). Mouse blood was washed and labeled with the DNA dye immediately after bleeding, whereas infected bovine blood was packed in ice and air freighted from Brisbane to Melbourne where it was washed twice at 4 C in phosphate buffered saline (PBS: bovine tonicity, 20 m&f sodium phosphate, 0.125 M NaCl, pH 7.3; mouse tonicity, 20 mM sodium phosphate, 0.149 M NaCl pH 7.3), then stored overnight at 4 C in PBS with 1 mg/ml added D(-)-glucose. The bovine blood was then rewashed twice before DNA labeling and cell sorting. The total time between bleeding the calves and DNA labeling never exceeded 25 hr. Preliminary experiments established that storage of mouse blood infected with B. rodhaini under the same conditions for 24 hr caused less than 5% loss of cells from the fluorescent cell peaks. Labeling cells with 33258 Hoechst. The 33258 Hoechst was obtained from Riedel-de Hadn AG, Seelze-Hannover and was used as described previously (Howard et al. 1979). Red blood cells were washed three or four times in mouse or bovine tonicity PBS as appropriate and the buffy coat was removed by aspiration. In some experiments the leukocyte contamination of mouse blood was reduced further (to less than
Babesia
spp.:SORTING OF INFECTED
0.05% of the total cells) by passage of the cells through a sulfoethyl cellulose column (Howard& al., 1978), although depletion of leukocytes was subsequently shown to be unnecessary. Washed red cells, 6 x 107, were resuspended in Eisen’s balanced salt solution [EBSS: Tris 5 m&f, sodium phosphate 5 n&f, MgSO, 1 mM, CaCl, 2 mM, KC1 6 mA4, with either 0.15 M NaCl (mouse tonicity) or 0.125 M NaCl (bovine tonicity)], plus 1 mg/ml D( -)-glucose, followed by addition of a sample of 100 or 200 pM 33258 Hoechst in EBSS plus glucose to the desired final dye concentration in 10ml total volume. Samples were immediately placed in darkness and incubated at 37 C in a humidified atmosphere of 10% CO, in air for 2 hr. At 37 C the dye enters living cells and binds reversibly to nuclear DNA (Arndt-Jovin and Jovin, 1977). The cells were then rapidly washed three times in PBS at 4 C in darkness, resuspended finally in 10ml, and sorted at this concentration. Cells were stored and sorted at 4 C in darkness. Cell sorting and analysis. The details of operation of the Becton Dickinson FACS II fluorescence activated cell sorter (FACS) and method of separation of fluorescent light from scattered laser light by optical filters have been described elsewhere (Howard and Battye, in press). Cells were sorted on the basis of their integrated low angle light scatter (approx. 1- 15”) and characteristic 33258 Hoechst fluorescence (350-nm excitation maximum and 460-nm emission maximum). The values of both parameters were expressed in 256 channel memory segments of the FACS II. The sensitivity of cell fluorescence detection could be increased by increasing the photomultiplier voltage or pulse amplifier gain setting. The sorted stream was directed into a siliconized glass tube containing 1 to 2 ml of PBS with 10% (v/v) fetal calf serum. Cells were collected by centrifugation (300g x 7 min, 4 C) and resuspended in 100 ~1 of the same medium with a fine bore Pasteur
RED CELLS
423
pipet. A drop of the suspension (lo-50 ~1) was applied to a glass slide, air dried, and stained with Giemsa’s stain after fixation in 90% (v/v) methanol. Infectivity studies. The effect of 33258 Hoechst labeling and sorting on the infectivity of blood cells with B. rodhaini was tested by the following qualitative method. Between 3 x 10” and 9 x 10” cells were sorted from the center of each of the fluorescent peaks Pl, P2, and P3 (Table II) and also from the nonfluorescent peak, diluted in PBS and lo* cells injected directly (iv) into BALB/c mice. As the steps prior to injection were performed on ice, the dye would have remained bound to parasite DNA (Howard et al. 1979). Several days after injection smears of peripheral blood from these mice were Giemsa stained and examined by light microscopy in order to verify the infectivity of injected parasites. RESULTS
Cell Sorting on the Basis of Low Angle Light Scatter The low angle scatter distributions of red cells from normal mouse or calf blood occasionally differed from the samples infected with Babesia spp. However, the differences were not reproducible, and generally too small to be exploited as a parameter for the sorting of different red blood cell populations . The low angle scatter distribution of B. rodhaini-infected blood depleted of leukocytes by repeated washing and aspiration of the buffy coat is compared in Fig. 1 with the scatter distribution of blood taken into Alsever’s solution and sorted directly without any attempt to remove leukocytes. Buffy coat removal effectively removed the leukocytes which are characterized by a higher low angle scatter intensity than red cells. In order to restrict fluorescence analysis to red blood cells and exclude smaller particles such as free parasites and parasite nu-
424
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species. In general, at the lowest dye concentrations tested the proportion of total cells which was fluorescent was much less than the degree of parasitemia, and, as the concentration was raised the proportion of fluorescent cells increased and the intensity of each fluorescent cell peak also increased. By using 100 pM dye for B. rodblood the haini and B. bovis-infected number of fluorescent cells was maximal, the fluorescence intensity of each of the fluorescent cell peaks was maximal and the resolution of the different peaks was also 0 optimal (Figs. 2A and C). The fluorescent 0 64 I20 192 256 profiles for B. bigemina-infected blood difRELATIVE LOU ANGLE SCATTER FIG. 1. The effect of leukocyte depletion on the low fered in that two fluorescent cell peaks angle scatter distribution of mouse blood infected with were clearly resolved at l-10 pM dye but Babesia rodhaini. Infected blood of 48% parasitemia at higher dye concentrations the two peaks was either depleted ofleukocytes by five washes in PBS merged into a broad peak centered at a and aspiration of the buffy coat (A), or, mixed directly with Alsever’s solution (B) prior to the analysis of low fluorescence intensity slightly less than the angle scatter. most fluorescent peak (Fig. 2B). The number of fluorescent cells was identical clei, upper and lower limits were set to the over the range of dye concentration tested for B. bigemina. A dye concentration of 2 intensity of low angle scattered light. pM was chosen for subsequent experiDual Parameter Cell Sorting ments with B. bigemina in order to resolve The effects of different dye concentra- the maximum number of different cell tions on the fluorescent cell distribution are types. Normal calf or mouse blood which had summarized in Fig. 2 for each of the Babesia
4
C
2 ii03 -’
; ,,‘. . L.. -. .;, : *w...__-
- .'., 2 .,.. \'..-plCw. i 'i 2
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0 64 128 192 256 RELATIVE FLUORESCENCE
‘. . .
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'-a 0 64 126 192 256 RELATIVE FLUORESCENCE
- ,.::. _.- L_"
-’ _
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a 0 64 I28 192 256 RELATIVE FLUOAESCEMCE
FIG. 2. Comparison of the fluorescent cell profiles for bloods infected with Babesia rodhaini, Babesia bigemina, and Babesia bovis after labeling with different concentrations of 33258 Hoechst (PM dye concentrations indicated in figure). (A) B. rodhaini-infected mouse blood (84% parasitemia) labeled with 0.1 - 100 @4 dye. (B) B. bigemina-infected calf blood (2.5% parasitemia) labeled with 2-100 FM dye. (C) E. bovis-infected calf blood (5.4% parasitemia) labeled with 2-100 pM dye.
Babesia
spp.: SORTING OF INFECTED
RED CELLS
425
TABLE I Comparison of the Proportion of Fluorescent Cells Labeled by 33258 Hoechst with the Parasitemia Determined by Light Microscopy
Parasite Babesia rodhaini
Sample 1
2 Babesia bovis
1
Babesia bigemina
2 1 2
Percentage fluorescent cells”
Percentage of cells parasitized (light microscopy)”
55.3 12.2 4.9 0.6’ 5.5 0.13’
49 10 4 channel 11 for B. rodhaini and > channel 25 for B. bovis and B. bigemina.
Ir Thin smears of infected blood were Giemsa stained and 1000 cells examined for parasites in several fields. ’ The fluorescent cell distributions for these samples of very low parasitemia were identical to those of higher parasitemia.
been leukocyte depleted was incubated at the dye concentrations chosen for optimal labeling of infected blood. Less than 0.1% of the total cells were fluorescent. Infected blood which had not been treated with the dye was also nonfluorescent (~0.1% fluorescent cells). The percentage of parasitized cells determined by light microscopy was almost identical to the percentage of fluorescent cells under optimal labeling conditions (Table I). The consistent finding that the percentage parasitemia determined by light microscopy was less than the percentage of fluorescent cells (Table I) may have been due to difficulty in identifying small stained Babesia parasites. Cells were sorted from the center of each peak in the fluorescent cell distribution and examined by light microscopy after Giemsa staining. The fluorescence properties of each of the selected populations are shown in Fig. 3 for each Babesia infection. Table II summarizes the light microscopy analysis of these populations. B. r&z&i-infected blood usually exhibited four fluorescent cell peaks in addition to the nonfluorescent cells (Fig. 3A). The fluorescent peaks are denoted Pl, P2, and P3 in Table II in order of increasing fluorescence intensity. The ratio of the fluores-
cence intensities of the maxima of each of the peaks Pl to P3 was 1.0:2.0:3.0 (Fig. 3A). As saturation levels of dye bound to DNA had been reached (Fig. 2A) this ratio
0 64 256 I211 192 RELATIVE FLUOflESCLWCE
FIG. 3. Selection of fluorescent cell peaks for sorting after optimal conditions of cell labeling. (A) Babesia rodhaini-infected mouse blood (79% parasitemia) labeled with 100 KM dye. With photomultiplier voltage (PV) of 575 and gain of 0.5, the fluorescent channels selected for sorting Pl, P2, and P3 were, respectively, 29-51.67-93, and 108-137, as indicated. (B) Babesia bovis-infected calf blood (4.3% parasitemia) labeled with 100FM dye. The PV was 600 and gain 0.5. The fluorescent channels selected for sorting Pl (32-85) are indicated. (C) Babesia bigemina-infected calf blood (1.2% parasitemia) labeled with 2 pM dye. The fluorescent channels selected for sorting Pl (56-96) and P2 (112-184) are shown for sorting under the same conditions as B.
426
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TABLE II Analysis of Red Cells from Babesia-Infected Blood Sorted on the Basis of DNA Content” Percentage of sorted cells
Parasite Babrsia
rodhaini
Babesia bovis
Babesia bigemina
Number of parasite nuclei per red cell
Sorted cells (channel limits) Nonfluorescent Fluorescent Pl” P21’ P3” Nonfluorescent Fluorescent Pl Nonfluorescent Fluorescent Pl P2
0
1
2
(O-3)
100
0
0
0
(29-51) (67-93) (108- 137) (O-5)
5 1 0 100
92 26 5 0
3 60 9 0
0 13 86 0
(32-85) (O-5)
1 100
94 0
5 0
0 0
2 0
96 81
2 19
0 0
(56-96) (112-184)
>3
” Cells from infected blood labeled under the same conditions as in Fig. 3. Cells, lo’- lo’, were sorted from the center of each peak (between the designated channel limits), methanol fixed on glass slides and examined by light microscopy after Giemsa staining. Six hundred cells were analyzed for each sample. ’ Smears of each of these sorted fluorescent cell populations indicated a significant proportion of parasites to be extracellular (2- 15% of the parasites in the sorted population in different experiments).
suggested that the DNA content of infected cells increased by integral amounts, i.e., integral numbers of parasites. No infected cells were seen in the nonfluorescent fraction. PI of B. rodhaini-infected blood consisted predominantly of red cells with a single parasite, whereas 60% of cells in P2 contained two parasite nuclei. The majority of the binucleate cells in P2 (>90%) contained two separate parasites. The cells with a3 nuclei in P3 contained morphologically diverse parasites, but approximately 80% of these infected cells contained 3 parasites each with one nucleus. Both P2 and P3 contained multinucleate parasites, but these occurred infrequently. The maximum number of parasites seen in a single cell was 8. Samples of B. rodhainiinfected blood of low parasitemia (~10%) contained a much lower proportion of fluorescent cells in the highly fluorescent peaks (aP3). B. bovis-infected blood (Fig. 3B) had a relatively simple distribution of fluorescent cells compared with B. rodhaini (Fig. 3A) exhibiting a single broad fluorescent peak
(Pl in Table II). This peak consisted of >90% singly infected cells with a small proportion of cells containing two parasites (Table II). After labeling B. bigemina-infected blood with 2 ,uM dye we sorted and examined the separate peaks (Fig. 3C). The peak of lowest fluorescence intensity (Pl in Table II) contained >95% singly infected red cells. The second peak (P2) was also predominantly singly infected cells, but in addition contained a significant proportion of cells with two parasites (5-20% in different samples (Table II)). The singly infected cells in PI and P2 were morphologically indistinguishable by light microscopy. The nonfluorescent cells consisted exclusively of uninfected red cells in both of the bovine infections (Table II). Infectivity
of Sorted Cells from
Babesia
rodhaini Infection Following injection of IO’ cells from Pl, P2, or P3 (4 mice per group), all animals died of B. rodhaini infection after IO-13 days. Injection of IO’ cells from the
Eobesia spp.: SORTING OF INFECTED
nonfluorescent peak also resulted in lethal B. rodhuini infection in a proportion of mice (death at 13- 14 days of l/4 and 214mice in two experiments). By examination of the fluorescence distribution of B. rodhuini-infected blood at very high sensitivity (Fig. 4) we could identify a minor population of cells with very low fluorescence which overlapped the nonfluorescent cell peak. Light microscopy analysis of cells sorted from the low fluorescence peak indicated that all of these cells were red cells which stained blue after Giemsa staining (and may therefore be reticulocytes), whereas cells sorted from channels O-15 of the nonfluorescent peak consisted of erythrocytes. Furthermore, approximately 5% of the cells in the low fluorescence peak contained a single very small parasite (red nucleus and very small ring of blue cytoplasm). Parasites were not detected in the sorted nonfluorescent cells. Injection of lo* cells sorted from the low fluorescence peak (Fig. 4) resulted in Babesia rodhaini infection and death at 14- 16 days of 3/4 mice in two experiments. However, infective particles were still 4r
:.
L 0
I
64
I
1
128 192 RELATIVE FLUORESCENCE
I 256
FIG. 4. Identitication of a population of Babesia rodhaini-infected cells which are poorly resolved from uninfected cells after 33258 Hoechst labeling. Infected blood of 53% parasitemia was labeled with 1OOpMdye and the fluorescent cell distribution examined with a PV of 625 and gain of 1.0. Under these conditions of increased sensitivity, nonfluorescent cells were sorted from channels 0- 15 and a population of cells with very low fluorescence was sorted from channels 36-62. Cells containing one parasite nucleus, comparable to the peak of lowest fluorescence intensity in Fig. 3A, are shown between channels 192and 256 in this figure.
RED CELLS
427
present in the nonfluorescent peak, as injection of lo* cells sorted from channels 0- 15 (Fig. 4) also produced infection in 2/4 mice (two experiments). These mice did not die until 18-20 days after injection. DISCUSSION
Cells infected with Babesiu spp. can be separated from uninfected cells and from each other on the basis of their DNA content after staining with 33258 Hoechst, thereby allowing a new method for the physical separation of red blood cells from infected blood. The essential features of this method are as follows: (1) preliminary purification of red cells by leukocyte depletion and washing (Fig. l), (2) incubation of red blood cells with the dye at 37 C. The concentration of dye used is critical in determining the final proportion of fluorescent cells and fluorescent cell profile (Howard et al. 1979; and Fig. 2). Provided the cells are maintained on ice and in darkness the dye remains bound to DNA for at least several hours (Arndt-Jovin and Jovin 1977; Howard et al. 1979); (3) cell sorting on the FACS using both low angle scatter and fluorescence parameters to restrict analysis to red cells. The dye can be removed from sorted cells by incubation at 37 C (Arndt-Jovin and Jovin, 1977). Some parasite viability is retained even when the dye is not removed before inoculation of the host. The concentration of 33258 Hoechst required for maximal fluorescence intensity for each peak in the distribution was 100 pM for each of the three Bubesiu species (Fig. 2), although this concentration was not chosen for separation of the cell types in B. bigeminu-infected blood. Two cell populations in B. bigeminu-infected blood were resolved at lower dye concentrations (2-10 pM) but merged at the higher saturating dye concentration. In contrast, maximal fluorescence intensity for each fluorescent cell peak in P. berghei-infected blood was achieved with 2 ~(LM dye in the
428
HOWARD
AND
same incubation medium (Howard et al. 1979). The morphology of Babesia varies, and there is debate as to the exact developmental sequence in the blood (Riek 1964, 1966; Holbrook et al. 1968; Kreier et al. 1975; Potgieter, 1977; Rudzinska and Trager, 1977). Light and electron microscope studies by these workers suggest explanations for the different populations of infected cells sorted on the basis of DNA content in the present work. B. rodhaini-infected cells were sorted into at least three fluorescent cell peaks (Fig. 3A). The fluorescence intensity of the center of each of these fluorescent peaks increased in an exactly integral ratio. Examination of the cells in Pl, P2, and P3 (Table II) by light microscopy confirmed that the majority of infected cells in these peaks contained, in order 1, 2, or 3 parasites. It is possible that a proportion of cells with fluorescence aP3 contained parasites derived from repeated merozoite invasion of a single red cell. The extremely high parasitemia of the B. rodhaini samples examined (30-80%) may have led to multiple invasion. Cytofluorimetric analysis of the DNA content of Plasmodium berghei merozoites and intracellular parasites (Bahr and Mikel, 1972), suggested that the DNA content of the merozoite was half that of young trophozoite forms. Although it could be concluded on this basis that the parasite DNA is very rapidly replicated after merozoite entry, the limits of sensitivity of the technique used plus the small amount of parasite DNA preclude a definitive conclusion. Biosynthetic labeling studies by Gutteridge and Trigg (1971) indicated that DNA synthesis in Plasmodium spp. occurs during the immature and mature trophozoite stages prior to schizogony and therefore precedes cytoplasmic and nuclear division. We observed a small number of B. rodhainiinfected cells with up to eight parasite nuclei in the third fluorescent peak (P3). The
RODWELL
DNA content of the merozoite (i.e., the parasite in mature heavily infected cells) may therefore be less than that of singly infected cells in PI. Infected cells were also identified in P2 with only a single parasite nucleus yet twice the fluorescence of uninucleated cells in Pl (Table II). These results suggest that the DNA content of Babesiainfected cells may also increase without cytoplasmic division. Other methods will be required to establish the exact time sequence of DNA synthesis between merozoite invasion and nuclear division. It is possible that the small parasite forms seen in some of the very weakly fluorescent reticulocytes (Fig. 4) represent recently invaded merozoites. As these parasiteinfected cells were no more fluorescent than the reticulocytes, their DNA would appear to be refractory to 33258 Hoechst labeling under the usual labeling conditions. At least some of these parasite forms must be viable, as inoculation of cells from the weakly fluorescent peak, and the nonfluorescent cells which overlap this peak infected mice. Sorting on the basis of low angle light scatter excluded the possibility that the viable particles in this fraction were merozoites. This result was unexpected in view of the apparent absence of parasites as judged by light microscopy of the nonfluorescent cells (Table II). Our experiments with P. berghei-infected blood sorted after 33258 Hoechst staining (Howard and Battye, in press; Howard et al. 1979) demonstrated that even when 4 x lo5 nonfluorescent cells were injected per mouse, P. berghei infection did not result. Fluorescent cells did, on the other hand, cause lethal P. berghei infection upon injection. With 100 pM 33258 Hoechst, B. bigemina-infected blood and B. bovisinfected blood each contained only one major fluorescent cell population which consisted predominantly of singly infected cells (Table II). The presence of a small number of cells containing two parasites in
Babesia spp.: SORTING
OF INFECTED
this fluorescent peak indicates that the DNA content of the singly infected cells was equivalent to at least two merozoites. These parasites are thought to multiply by a budding process to produce one pair of pyriform parasites which leave the cell to reinvade other red cells (Riek, 1966). The cells containing two parasites in this fluorescent peak would therefore be mature infected cells about to rupture. The budding process of Babesia differs from schizogony typical of Plasmodium in that nuclear division is completed at a very late stage of merozoite development, with little if any nuclear material remaining in the remnant of the mother cell (Potgieter, 1977). By contrast, after completion of schizogony in Plasmodium spp., considerable nuclear material remains in the residual parasite body (Aikawa, 1966). Infected cells containing parasites with less DNA than the major fluorescent peak (i.e., equivalent to a merozoite) were not seen. This suggests that rapid doubling of the parasite DNA content after red cell invasion may be a common feature of Babesia and Plasmodium infections. Another common feature might be the delay between DNA replication and nuclear and cytoplasmic division of the parasite. Very few red blood cells from B. bovis or B. bigemina-infected blood had fluorescence greater than the major fluorescent peak. Although these highly fluorescent cells were not examined by light microscopy, they may have contained three or four parasites as a result of multiple infection. One unusual feature of the fluorescent cell profile for B. bigemina-infected blood was the resolution of two infected cell populations at dye concentrations less than the saturation level (Fig. 2B). Light microscopy analysis of the two populations failed to indicate significant differences in gross morphology of these parasites after Giemsa staining. However, the greater prevalence of cells with two parasites in the more highly fluorescent peak was a consistent
RED
CELLS
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difference (Table II). These two cell populations appear to have different “affinities” for 33258 Hoechst such that after incubation in 2 pM dye for 2 hr they bound different amounts of dye. Incubation in 100 pM dye for the same period resulted in saturation binding for both populations. The slight decrease in fluorescence intensity of the P2 cells seen after treatment with 100 pM dye, compared to their fluorescence intensity after 2 pM dye (Fig. 2B), may be similar to the drop in fluorescence intensity of P. berghei-infected cells at supraoptimal dye concentrations (Howard et al. 1979) a phenomenon also observed when isolated DNA is labeled with high concentrations of the dye (Latt and Stetten 1976). The cell sorting method yields 99-100% pure uninfected cells from Babes&infected blood and 90% pure infected cells. Approximately 1.5 x lo6 uninfected cells, and 3-5 x lo” infected cells from the center of each fluorescent peak, can be sorted per hour with a 50% parasitemia of B. rodhaini. The number of infected cells collected per hour is proportionally fewer for B. bovis and B. bigemina-infected samples in which parasitemias reach a maximum of lo- 15%. These cell numbers are sufficient for microbiochemical analyses. However the method cannot resolve all of the morphological stages seen in infected blood samples. With B. bigemina-infected blood, at least 15 morphological forms of infected cells were described by light microscopy (Riek 1964). Separation on the basis of DNA content fails to resolve some cells of identical DNA content with obviously different morphologies-for example, uninucleate trophozoite-infected cells and cells containing two merozoites (Table II). Further advances in flow systems technology such as sorting on the basis of DNA content and a parameter such as high angle light scatter which is related to complex aspects of internal cell architecture, may resolve additional subpopulations of infected cells. The present method is being used to
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separate uninfected and infected cells from bovis-infected blood after radioiodination of cell surface proteins to enable pathological and immunological studies.
Babesia
ACKNOWLEDGMENTS
erinary Research
This work was supported by the UNDPiWorld Bank/WHO Special Program for Research and Training in Tropical Diseases and by the Australian National Health and Medical Research Council. The expert assistance of Dr. F. Battye in operating the FACS is gratefully acknowledged. The advice of Drs. L. L. Callow and G. F. Mitchell in preparing the manuscript is greatly appreciated. We are also grateful to Joan Curtis for technical assistance.
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