- Email: [email protected]
Theileria parva: Effects of irradiation on a culture of parasitized bovine lymphoid cells
EXPERIMENTAL PARASITOLOGY 38, 64-74
Theileria
(1975)
on a Culture parva: Effects of Irradiation of Parasitized Bovine Lymphoid Cells
A. D. IRVIN,~ C. G. D. BROWN,~ AND D. A. STAGC s VNDP/FAO
Tick-borne Organization, (Accepted
Diseases Project, East African Veterinary Muguga, P.O. Box 32, Kikuyu, Kenya for publication
October
Research
28, 1974)
IRVIN, A. D., BROWN, C. G. D., AND STAGG, D. A. 1975. Theileria parva: Effects of irradiation on a culture of parasitized bovine lymphoid cells. Experimental Parasitology 38, 64-74. Aliquots of a culture of Thderia pawa-infected bovine lymphoid cells were irradiated at 0, 300, 600, 900, and 1200 rads. The short-term effects of irradiation were evaluated on examination of Giemsa-stained smears and on autoradiography of cells labeled with [3H]thymidine. Irradiation inhibited cell division but parasite division did not appear to be inhibited and macroschizont nuclear particles increased in number, frequently to several hundred per schizont. There was no evidence of an increased percentage switch from macro- to microschizont. Apparently viable cells were still present in all cultures 4 days after irradiation. INDEX DESCRIPTORS: Theileria parva; Bovine lymphoid cells; Irradiation; [“HIThymidine; Autoradiography; Tissue culture; Macroschizont; Cell lines.
possible means of immunizing cattle. Attention has now been turned, therefore, toward the possibility of using T. parvuinfected bovine lymphoid cells irradiated in cell culture, as a means of immunization. This paper is a preliminary study to this work and examines the short-term effects of irradiation on a parasitized cell culture.
The possibilities of attenuating Theileria paroa, the causative agent of East Coast fever (ECF) of cattle, by irradiation, have previously been examined by Cunningham et al. (1973) and Purnell et al. (1974). The former authors irradiated infective particles harvested from the tick vector ( Rhipicephulus uppendiculatus), and the latter authors irradiated infected ticks. The effects of irradiation on these preparations were then assessed on their ability to infect groups of susceptible cattle with ECF, or else protect them against the disease. Neither group of workers considered their methods had practical application as
MATERIALS
Cell Culture The culture was a T. parva-infected bovine lymphoid cell line (designated C2), passage 35, grown in 250 ml Falcon Flasks 4 as a static cell culture (Malmquist, Nyindo, and Brown 1970). The medium used was Eagle’s MEM supplemented with 2070 foetal calf serum. The initial culture was seeded at 3 x lo5 cells/ml and after 24 hr divided into 5 x 50 ml aliquots each con-
1 On Overseas Development Administration seeondment from the ARC Institute for Research on Animal Diseases, Compton, Berkshire (Research Project R2845). * FAO Staff assigned to the Project. 3 On Overseas Development Administration secondment from the Central Veterinary Laboratory, Weybridge, Surrey (Research Project R2845).
4 Falcon 64
Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
Plastics,
Oxnard,
California,
USA.
IRRADIATION EFFETE ON Theileria--~0s~ taining approximately 5 X lo” cells/ml of medium. These aliquots were then irradiated at the following five doses: 0, 300, 600, 900, and 1200 rads, using a 6oCo source with an output of 2.13 krad/min. Immediately after irradiation each 50 ml aliquot was divided into 8 x 6 ml aliquots, each of which was placed in a 25 ml Falcon flask; the cells still being suspended in the original medium (i.e., a total of 5 x 8 flasks each holding 6 ml of cell culture). Each group of eight flasks represented four pairs to be examined at daily intervals starting 24 hr after the final division lof the culture. Twenty-four hours prior to examination of pairs of flasks, [6-3H]thymidine ( sp act 5 Ci/mmole) was added to one of each pair at the rate of 0.25 &i/ml of medium. Except for the addition of thymidine, labeled and control prepa’rations were handled and treated identically. Cell Examinations Cell counts were performed on each culture using a haemocytometer. Percentage of viable cells was determined concurrently using nigrosin stain to detect dead cells ( Kaltenbach, Kaltenbach, and Lyons 1958). Cultures for smear preparation were centrifuged for 5 min at 700g. Cells were then washed in phosphate buffered saline; centrifugation and washing were repeated and the cells were resuspended in growth medium before a final centrifugation. Smears were prepared from the resultant cell pellet. Those for autoradiography were fixed in methanol but left unstained; the remander were fixed as above and stained in 10% Giemsa stain. Giemsa-stained smears were prepared from both labeled and unlabeled cultures, and the following parameters determined from cell counts and examinations: (a) Percentage of cells containing parasites. (b) Percentage of multinucleate cells. (c) Percentage of cells in mitosis (mitotic index).
caL
cumwm
65
(d) Percentage of cells in which the macroschizont stage of the parasite (normally present in parasitized bovine lymphoid cells grown in cul’ture) had transformed to the microschizont stage. Parameters (a)-(d) were determined from counts of 500 cells on each slide. (e) The mean number of nuclear particles (mean schizont number-MSN) in 50 intracellular macroschizonts per slide. The problems involved in such counts and the degree of accuracy expected have been previously discussed (Bamett, Brocklesby, and Vidler 1961) . Base values for parameters (a)-(e) were determined from a cell smear made prior to irradiation and culture division. (f) The size range of 100 intact, circular, mononucleate, parasitized cells. This was determined on examination of smears under a 100x oil immersion objective with 8x eyepieces, one of which contained a graticule marked into 100 equal divisions. Each cell was assigned to a size category based on graticule divisions (510, 10-15, 15-20, 20-25, 25-30, 30-35), and in this way a histogram of size range was constructed. Each graticule division represented 1.25 pm; actual size of cells could therefore be readily calculated. All smears were prepared and examined by the same person, and the same part of the smear examined on each slide. Autoradiography Smears for autoradiography were extracted in 5% aqueous trichloracetic acid for * hr at 4 C, and then processed as previously described (Irvin et al. 1974), using a 6-day exposure period. The following further parameters were determined from the examination of 500 cells per smear: (g) Percentage of unlabeled cells. (h) Percentage of cells with labeled nuclei.
66 (i) Percentage parasites.
IRVIN,
of
cells
with
BROWN
labeled
AND
STAGG
107 I
RESULTS
Viable Cell Count The division of cell cultures was calculated to yield a final cell concentration per 6 ml aliquot, of 5 x lo” cells per ml. However, following the various manipulations the number of viable cells fell to 3.6 x lo6 cells per ml; this was taken as the base value of cells in culture (Fig. 1). The figure also shows the changes in number of viable cells up to 4 days after irradiation. Nonirradiated cultures showed a regular increase in cell number up to Day 3, followed by a fall. Extrapolation based on Fig. 1 showed that nonirradiated culture had a mean doubling time of 28 hr. The nonirradiated culture that had [ sH] thymidine added showed a slight initial fall, but subsequently the growth rate paralleled that of the other nonirradiated culture at a lower level. Cultures irradiated at 300 rads showed a slight fall in number of viable cells up to Day 3, but then a marked rise with cell growth rate similar to that observed in nonirradiated cultures between Days 1 and 3. The [ 3H]thymidine labeled culture showed a similar growth pattern to the nonlabeled culture, but again at a lower level. Above 300 rads of irradiation no significant difference could be detected in viable cell counts between cultures with or without [3H] thymidine, the two values were therefore combined and the r.esultant mean plotted in Fig. 1. Cultures irradiated at 600, 900, and 1200 rads maintained a fairly constant viable cell number up to Day 2, then all fell sharply, the rate of fall being proportional to the amount of irradiation. Even at 1200 rads approximately lo4 cells were apparently viable after 4 days. Percentage of Cells Containing
Parasites
96.6% of cells in the preirradiation control smear were parasitized with macro-
1
2
4
3
Day.5
FIG. 1. Daily cell numbers/ml in Theileriu parua cultures irradiated at five different levels ( * = cultures labeled with [3H] thymidine).
schizonts of T. paroa. Subsequent smears showed a range from 95.6 to 99.470, but no significant differences as a result of different treatments, could be detected. Percentage of Multinucleate
Cells
6.2% of cells in the preirradiation control smear contained more than one nucleus. The range in the other smears was 0.8-10.470. In the nonirradiated cultures there was a slight fall, with time, in the percentage of multinucleate cells, and the lowest value was reached on Day 3. In the other cultures the highest values were usually found on Day 2, but no specific trend could be demonstrated, nor could any difference be detected between labeled and nonlabeled cultures. Percentage of Cells in Mitosis The mitotic index of the control smear was 2.870. The values for the other smears are shown in Fig. 2. No difference could be detected between labeled and unlabeled cultures so the values were combined and the means plotted in the figure. On Day 1 all cultures showed a similar value around 2%. The nonirradiated cultures maintained this value throughout the experimental pe-
IRRADIATION
EFFECI’S
ON
~hd!dU-HOST
riod, but all the others showed a marked rise up to a peak at Day 3, this rise being proportional to the amount of irradiation delivered. After Day 3 there was a sudden fall in the mitotic index in all cultures.
CELL
67
CULTURE
6or
Percentage of Cells Containing Microschizonts Microschizonts were detected intermittently in all cultures up to the end of Day 3, but never exceeded 0.4%. The value for the control smear was 0.270. Mean Schizont Nuclear Number (MSN) The MSN of the control smear was 13.4. The values for the other smears are shown in Fig. 3. In the unlabeled nonirradiated cultures there was a gradual fall in MSN after a slight initial rise. The labeled nonirradiated culture showed a similar pattern, but at a consistently higher level. The 300 rad group of cultures showed a pronounced initial rise and subsequent fall, and again the labeled cultures showed consistently higher values than the unlabeled ones. At 600 rads and above it was not possible to detect differences between labeled and unlabeled cultures, the two values were therefore combined and the means plotted in Fig. 3. The MSNs in the 600 rad cultures reached a peak on Day 2 and in the 900 and 1200 rad cultures reached peak on Day 3; following peak there was a rapid fall in MSN counts of all cultures. The peak 30 1
.c
4 1
2 Days
3
4
FIG. 2. Daily mitotic indices in Theileriu parua cultures irradiated in five different levels.
1
I 1
2
3
4
Days
FIG. 3. Daily values of mean schizont nuclear number in Theileria parua cultures irradiated at five different levels ( * = cultures labeled with L3H]thymidine).
values were in all cases proportional to the amount of irradiation delivered to the cultures. Cell Size The size range of cells is shown in Fig. 4. On Day 1 no significant difference was detectable, but on Day 2 there was an incr.easing percentage of large cells proportional to the amount of irradiation, this resulted in a shift of the histogram to the right at higher irradiation doses. This shift was most pronounced on Day 3, it was also generally greater in labeled than unlabeled cultures. The range of cell size is shown by the amount of horizontal spread of the histogram. This spread became more marked, with time, at higher irradiation levels, but in nonirradiated cultures the spread became less. Autoradiography The percentage of cells with labeled nuclei is shown in Fig. 5. The fall in per-
68
IRVIN,
BROWN
AND
% Controls
STACG
w 3HT Cultures
Rodiation
dose (rads)
..-
o-
10
I
20
30
0 10 Days parva
centage was proportional to the amount of irradiation to which cultures were exposed. Nonirradiated cultures maintained 100 r
80 . 70. 60 % 50 40. 30. 20 10
,
I
1
2 Days
3
30
3
2
FIG. 4. Size ranges of cells in Theileria (Each graticule division = 1.25 ,um).
t
20
600
3
FIG. 5. Percentage of cultured bovine lymphoid cells with [3H]thymidine incorporated into nuclei, following irradiation at five different levels.
cultures
irradiated
at five
different
levels.
a constant level until Day 3, but then fell. Irradiated cultures showed only a slight fall in percentage of labeled nuclei after 24 hr but then a rapid fall followed by recovery occurred. In the case of 600 and 900 rad cultures, the percentage reached a final level higher than that of nonirradiated cultures. The percentage of cells containing labeled parasites is shown in Fig. 6. In this case there was an increase in percentage proportional to the amount of irradiation to which cultures were exposed. No difference in [“Hlthymidine uptake between irradiated and nonirradiated cultures was detected until Day 2. The percentage of cells showing no label present, either in nucleus or parasite, was simply a reflection of the combined details recorded in Figs. 5 and 6, and no specific trends could be detected. The overall effect of irradiation on [“Hlthymidine uptake was that, with increasing doses of irradiation there was an increase in uptake
IRRADIATION
EFFECTS
ON
~hdf?h-HOST
CELL
CULTURE
69
of [3H]thymidine by the parasites but a decrease in uptake by the cell nuclei (Fig. 7). Parasite and Cell Morphology Figure 8 shows normal T. parva-infected bovine lymphoid cells, taken from the preirradiation control smear. In smears from cultures that had been irradiated the changes immediately noted were: the increase in cell size, the increase in mitotic index (with a majority of abrrormal mitoses), and the alteration in parasite morphology (Fig. 9). Macroschizont nuclear particles became smaller and increased in number, frequently to several hundred per schizont. In some cases cytomere-like blocks of schizont material were seen in infected cells (Fig. 10). Cultures examined 2-3 days after irradiation usually showed
%
FIG. 6. Percentage of cultured bovine lymphoid cells with [3H]thymidine incorporated into macroschizonts of Theileria paroa, following irradiation at five different levels.
FIG. 7. Autoradiograph of Theileria paruu-infected bovine lymphoid cells 2 days after irradiation at 900 rads. Culture was subjected to a 24-hr pulse of [‘Hlthymidine before harvesting. Counterstained with Giemsa X 1300. Three cells show normal nuclear labeling. In three other cells (arrowed) only the parasites are labeled; the largest of these cells is in mitosis and macroschizont nuclear particles are distributed among the nuclear segments of the cell.
70
IRVIN,
FIG. 8. Smear x1300.
of normal
Theileria
BROWN
AND
pawa-infected
two distinct populations of cells: those affected by irradiation undergoing the various morphological changes, and those of normal morphology, which were apparently unaffected by irradiation and were presumably destined to continue normal multiplication and repopulation of the culture. At higher doses of irradiation these appar.ently-normal cells were at a very low level, but even at 1200 rads some were continually present. DISCUSSION
The effects of irradiation on a cell culture system are very complex, and numerous physical factors relating to both the irradiation dose and the culture system have to be considered. Firstly the cells were nonsynchronized and the system examined here was further complicated by the presence of parasites in the cells, which themselves exert a profound influence on cell growth and behavior.
STAGG
bovine
lymphoid
cells.
Giemsa
stain
Cultures were examined for only 4 days, since beyond that time the accumulation of cell by-products and the exhaustion of growth metabolites would have provided further complicating factors in the interpretation of results. Even so, between Days 3 and 4, there was some evidence that these factors were becoming exerted, particularly in the nonirradiated cultures. Changing the medium could have circumvented these problems but, in itself, would have been a complicating factor. The addition of [ 3H]thymidine also altered the growth pattern of cells at 0 and 300 rads. At 600 rads and above, any effect of the addition of [ 3H]thymidine, was masked by the much greater effects of irradiation The nature of the [3H]thymidine effect was not examined, although its influence as an additional radiation source and as a DNA inhibitor could have both played some part (Evans 1966; Painter and Rasmussen 1964). Despite the complexity
IRRADIATION
EFFECXS
ON
%df&Z-HOST
CELL
CULTURE
71
FIG. 9. Smear of Theileriu parua-infected bovine lymphoid cells 3 days after irradiation at 1200 rads, showing irradiation giant cells, abnormal mitoses and increase in parasite mass. Giemsa stain X 1300.
of the system certain well-defined patterns emerged, and although the cell and parasite activities are closely integrated it is more convenient to consider separately their reactions to irradiation. Reaction of Cells to irradiation The general responses of mammalian cells to the effects of irradiation have been (Bond, reviewed by earlier workers Fliedner, and Archambeau 1965), whose descriptions have formed the basis of the interpretations of the current results. The initial handling procedures slightly depressed cell growth rate as shown by the nonirradiated cultures in the first 24 hr (Fig. 1). The initial fall in viable cell count of the irradiated cultures was probably partly due to handling but also as a result of death of cells in the very radiosensitive M and early S phases of the cell
cycle at the time of irradiation (Terasima and Tolmach 1963). Cells in the Gl and G2 phases could be expected to be less and consequently some radiosensitive would undergo normal division. This is shown by the slight rise in viable cell counts of irradiated cultures between Days 1 and 2. The fact that surviving cells were undergoing almost normal cell function for the first 24 hr, is further shown by the uptake of [ 3H] thymidine in their nuclei ( Fig. 5). This demonstrated that DNA synthesis was proceeding at a rate only slightly below that of nonirradiated cells. All cultures showed a similar slight fall in mitotic index at the end of the first day. No difference was noted between irradiated and nonirradiated cells since, although cells in mitosis would have been killed by irradiation, others would presumably have advanced along the cell cycle
72
IRVIN,
BROWN
FIG. 10. Smear of Theileria parua-infected at 1200 rads, showing two partially ruptured Giemsa stain X 1300.
AND
STAGG
bovine lymphoid cells 3 days after irradiation cells with cytomere-like blocks of macroschizonts.
to the M phase within the 24 hr following irradiation. Between Days 1 and 3 nonirradiated cultures were growing with an approximate doubling time of 28 hr ( Fig. 1) . During this time mitotic index and DNA synthesis remained more or less constant (Figs. 2, 5). After Day 3 however cells began to die, as shown by the falls in both viable cell count and uptake of [3H] thymidine by cell nuclei. This presumably occurred as a result of exhaustion of the cell medium. In the irradiated cultures the numbers of viable cells were still high at the end of Day 2 (Fig. 1) but many of these apparently viable cells were unable to synthesize DNA as shown by the rapid fall in percentage of labeled cells which started a day earlier (Fig. 5). The 300 rad culture had recovered from the effects of irradiation by the end of Day 3: DNA synthesis had returned to the normal level (Fig. 5) and, between Days 3 and 4, cell growth rate was similar to that observed in nonirradiated cultures between Days 1 and 3 (Fig. 1). The reason for this could have been that, in contrast to the nonirradiated cultures, the cell medium had not been exhausted by Day 3 (since earlier growth rate had been lower) and cells were able to multiply uninhibited.
Most cells which die following irradiation are capable of at least one division, and decreasing numbers can divide two, three or more times before death (Bond, Fliedner, and Archambeau 1965). This is demonstrated well (Fig. 1) for the viable cell counts of the 600, 900, and 1200 rad cultures; all fell steadily from Days 2 to 4 as increasing numbers of cells died in each culture. However, Fig. 5 shows that DNA synthesis was returning to normal levels on Day 3 in the 600 and 900 rad cultures, and on Day 4 in the 1200 rad cultures. In the 600 and 900 rad cultures the final levels recorded for percentage of labeled cells were, in fact, well above normal. This apparent revival of DNA synthesis may partly have represented cell recovery but also an accumulation of labeled cells which were unable to complete cell division. This is suggested by the rise in mitotic index proportional to irradiation (Fig. 2), indicating inability of cells to proceed beyond the M phase. In an earlier study Brown et al. (unpublished data) found that, although cells apparently survived irradiation above 600 rads, recovery of cultures only occurred at 600 rads and below. This was explained on the basis that cell numbers had fallen below the critical number of 4.3 x lo3 (Malmquist, Nyindo, and Brown 1970)
IRRADIATION
EFFECTS
ON
~heihia--HOST
and that toxic products had accumulated in the medium. There was an increasing incidence of radiation giant cells up to Day 3 proportional to the level of irradiation (Fig. 4). This presumably resulted from an increasing incidence of cells unable to divide but still able to grow in size. On Giemsa-stained smears, these giant cells were up to 4X the area of normal cells. Many giant cells were apparently unable to progress beyond the M phase, as shown by the increasing mitotic index related to irradiation dose and time. Most of the mitotic figures seen in giant cells appeared to be abnormal metaphases, and the mechanism responsible for further progression was, in many cases, apparently deficient. After Day 3 there was a general fall in the percentage of giant cells (Fig. 4) presumably due to cell death and lysis. This explains the fall in mitotic indices by Day 4 (Fig. 4), and further emphasizes the gen.eral recovery of cell cultures at this time. Reaction of Intracellular to Irradiation
Parasites
Under normal circumstances macroschizont and host cell activities are apparently synchronized, since the MSN of the macroschizonts does not appreciably alter with successive passages of culture (Hulliger, Brown, and Wilde 1966). Furthermore the fact that neither free macroschizonts nor nonparasitized cell can apparently grow on their own, suggests that each is dependent on the other. This interdependence however, is disrupted by irradiation, and parasite and host cell respond in different ways, the cell being more radiosensitive than the parasite. Purnell et al. (1974) have shown, for example, that T. parua in the tick, is unaffected by irradiation doses up to 10 krad and a decreasing proportion of parasites can survive doses up to 45 krad. Cell division was increasingly inhibited at higher irradiation doses. Parasite division appeared however to be unaffected
CELL
CULTURE
73
and in arrested cells the MSN sometimes became very high. The fact that the mean MSN was highest in the 1200 rad cultures (Fig. 3) may simply be a reflection of the fact that these cultures contained the highest percentage of arrested cells. Other agents or factors which arrest or retard cell division, such as colchicine (Hulliger 1965; Hulliger et al. 1964), actinomycin D (Moulton, Krauss, and Malmquist 1971) and raised incubation temperature (Hulliger, Brown, and Wilde 1966), have been shown to result in similar MSN increases, Two other possibilities must however be considered in relation to increased MSN following irradiation: firstly the parasite may have been directly stimulated by irradiation to undergo division; the increasing percentage of labeled parasites with higher doses of irradiation, and the observed morphological changes in the parasite suggested that there was increased and altered activity in the macroschizonts. Other work has shown, for example, that T. parva macroschizonts do not readily incorporate [ 3H] thymidine under normal conditions (Irvin et al. 1974; Moulton, Krauss, and Malmquist 1971). Secondly, the altered environment of the cell may have affected parasite behavior, since rapid nuclear division of macroschizonts and the formation of cytomere-like blocks (Fig. 10) have also been observed in bovine hamster heterokaryons parasitized with T. parva (Irvin et al. 1975). A comparison of the uptake of [ 3H]thymidine in colchicine-arrested cells (where the parasite is less likely to be directly affected by the cell inhibitor) with [3H]thymidine uptake in irradiated cells, might help to evaluate these questions. So far it has not been possible to irradiate free macroschizonts to examine the direct effects of irradiation on this stage of the parasite. There was no evidence in this work that irradiation speeded-up the switch of the parasite from macroschizont to microschizont, and, although many of the later
74
IRVIN, BROWN AND STAGG
schizonts contained large numbers of small particles, they morphologically resembled macroschizonts rather than microschizonts. However, Brown et al. (unpublished data) have shown that different passage levels ‘of culture show different propensities for microschizont formation, and in one .experiment these workers showed that irradiation did result in an increased incidence of microschizont formation. It has also been shown in other systems where cell division has been r.etarded that an increased percentage of microschizonts occurs (Hulliger, Brown, and Wilde 1966). Many factors need further investigation and, most important of all, the infectivity and possible attenuation of the parasites need to be evaluated by in viva studies. ACKNOWLEDGMENTS We are grateful to M. P. Cunningham and our colleagues on the Project for help and advice. Our thanks are also due to Professor G. Mugera for allowing us access to the O°Co source at the Faculty of Veterinary Science, University of Nairobi, and to R. C. Payne and Mrs. S. Stagg for assistance with photography and diagrams. The technical assistance of L. Njuguna is gratefully acknowledged. This paper is published by the kind permission of Dr. G. L. Corry, The Director, EAVRO. The Project is supported by the United Nations Development Programme, with the Food and Agriculture Organization of the United Nations as the Executing Agency, in cooperation with the East African Community. The Project is also supported by the Overseas Development Administration of the United Kingdom (Research Project R2845), the United States Department of Agriculture, the Rockefeller Foundati’on, the Intemational Atomic Energy Agency, and the Pfizer Corporation.
REFERENCES BARNETT, S. F., BROCKLESUY, D. W., AND VIDLER, B. 0. 1961. Studies on macroschizonts of Theileria parva. Research in Veterinary Science 2, 11-18. BOND, V. P., FLIEDNER, T. M., AND ARCHAMBEAU, J. 0. 1965. “Mammalian Radiation Lethality.” Academic Press, London. CUNNINGHAM, M. P., BROWN, C. G. D., BURRIDGE, M. J., MUSOKE, A. J., PURNELL, R. E., AND
DARGIE, J. D. 1973. East Coast fever of cattle : “Co irradiation of infective particles of 20, Theileria partia. Journal of Protozoology 298-300. EVANS, E. A. 1966. “Tritium and its Compounds.” Butterworths, London. HULLIGER, L. 1965. Cultivation of three species of Theileria in lymphoid cells in uitro. Journal of Protozoology 12, 649-655. HULLIGEH, L., BROWN, C. G. D., AXD WILDE, J. K. H. 1966. Transition of developmental stages of Theileria parva in vitro at high temperature. Nature (London) 211, 328-329. HULLIGER, L., WILDE, 1. K. H., BROWN, C. G. D., AND TURNER, L. 1964. Mode of multiplication of Theileria in cultures of bovine lymphocytic cells. Nature (London) 203, 728-730. IRVIN, A. D., BHOWN, C. G. D., BOARER, C. D. H., CRAWFORD, J. G., AND KANHAI, G. K. 1974. Autoradiographic evidence for the occurrrence of cell fusion in cultures of Theileria-infected bovine lymphoid cells. Research in Veterinary Science 16, 137-142. I~IVIN, A. D., BROWN, C. G. D., STAGG, D. A., KANHAI, G. K., AND ROWE, L. W. 197.5. Hybrid cells, infected with Theileria parua, formed by fusion of hamster and mouse cells with parasitized bovine lymphoid cells. Research in Veterinary Science, in press. KALTEXBACH, J. P., KALTENBACH, M. H., AND LYONS, W. B. 1958. Nigrosin as a dye for differentiating live and dead ascites cells. Experimental Cell Research 15, 112-117. MALMQUIST, W. A., NYINCO, M. B. A., AND BROWN, C. G. D. 1970. East Coast fever: Cultivation in zjitro of bovine spleen cells infected and transformed by Theileria parva. Tropical Animal Health and Production 2, 139-145. MOULTON, J. E., KRAUSS, H. H., AND MALMQUIST, W. A. 1971. Growth characteristics of Theileria puma-infected bovine lymphoblast cultures. American Journal of Veterinary Research 32, 1365-1370. PAINTER, R. B., AND RASMUSSEN, R. E. 1964. A pitfall of low specific activity radioactive thymidine. Nature (London) 201, 409-410. PURNELL, R. E., BROWN, C. G. D., BURRIDGE, M. J., CUNNINGHAM, M. P., EMU, H., IRVIN, A. D., LEDGER, M. A., NJUGUNA, L. M., PAYNE, R. C., AND RAULEY, D. E. 1974. ‘“Co irradiation of Theileria parua in its tick vector Rhipicephalus appendiculatus. International Journal for Parasitology 4, 504-511. TERASIMA, T. T., AND TOLMACH, L. J. 1963. Variation in several responses of Hela cells to X-irradiation during the division cycle. Biophysics Journal 3, 11-33.
Theileria
(1975)
on a Culture parva: Effects of Irradiation of Parasitized Bovine Lymphoid Cells
A. D. IRVIN,~ C. G. D. BROWN,~ AND D. A. STAGC s VNDP/FAO
Tick-borne Organization, (Accepted
Diseases Project, East African Veterinary Muguga, P.O. Box 32, Kikuyu, Kenya for publication
October
Research
28, 1974)
IRVIN, A. D., BROWN, C. G. D., AND STAGG, D. A. 1975. Theileria parva: Effects of irradiation on a culture of parasitized bovine lymphoid cells. Experimental Parasitology 38, 64-74. Aliquots of a culture of Thderia pawa-infected bovine lymphoid cells were irradiated at 0, 300, 600, 900, and 1200 rads. The short-term effects of irradiation were evaluated on examination of Giemsa-stained smears and on autoradiography of cells labeled with [3H]thymidine. Irradiation inhibited cell division but parasite division did not appear to be inhibited and macroschizont nuclear particles increased in number, frequently to several hundred per schizont. There was no evidence of an increased percentage switch from macro- to microschizont. Apparently viable cells were still present in all cultures 4 days after irradiation. INDEX DESCRIPTORS: Theileria parva; Bovine lymphoid cells; Irradiation; [“HIThymidine; Autoradiography; Tissue culture; Macroschizont; Cell lines.
possible means of immunizing cattle. Attention has now been turned, therefore, toward the possibility of using T. parvuinfected bovine lymphoid cells irradiated in cell culture, as a means of immunization. This paper is a preliminary study to this work and examines the short-term effects of irradiation on a parasitized cell culture.
The possibilities of attenuating Theileria paroa, the causative agent of East Coast fever (ECF) of cattle, by irradiation, have previously been examined by Cunningham et al. (1973) and Purnell et al. (1974). The former authors irradiated infective particles harvested from the tick vector ( Rhipicephulus uppendiculatus), and the latter authors irradiated infected ticks. The effects of irradiation on these preparations were then assessed on their ability to infect groups of susceptible cattle with ECF, or else protect them against the disease. Neither group of workers considered their methods had practical application as
MATERIALS
Cell Culture The culture was a T. parva-infected bovine lymphoid cell line (designated C2), passage 35, grown in 250 ml Falcon Flasks 4 as a static cell culture (Malmquist, Nyindo, and Brown 1970). The medium used was Eagle’s MEM supplemented with 2070 foetal calf serum. The initial culture was seeded at 3 x lo5 cells/ml and after 24 hr divided into 5 x 50 ml aliquots each con-
1 On Overseas Development Administration seeondment from the ARC Institute for Research on Animal Diseases, Compton, Berkshire (Research Project R2845). * FAO Staff assigned to the Project. 3 On Overseas Development Administration secondment from the Central Veterinary Laboratory, Weybridge, Surrey (Research Project R2845).
4 Falcon 64
Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
Plastics,
Oxnard,
California,
USA.
IRRADIATION EFFETE ON Theileria--~0s~ taining approximately 5 X lo” cells/ml of medium. These aliquots were then irradiated at the following five doses: 0, 300, 600, 900, and 1200 rads, using a 6oCo source with an output of 2.13 krad/min. Immediately after irradiation each 50 ml aliquot was divided into 8 x 6 ml aliquots, each of which was placed in a 25 ml Falcon flask; the cells still being suspended in the original medium (i.e., a total of 5 x 8 flasks each holding 6 ml of cell culture). Each group of eight flasks represented four pairs to be examined at daily intervals starting 24 hr after the final division lof the culture. Twenty-four hours prior to examination of pairs of flasks, [6-3H]thymidine ( sp act 5 Ci/mmole) was added to one of each pair at the rate of 0.25 &i/ml of medium. Except for the addition of thymidine, labeled and control prepa’rations were handled and treated identically. Cell Examinations Cell counts were performed on each culture using a haemocytometer. Percentage of viable cells was determined concurrently using nigrosin stain to detect dead cells ( Kaltenbach, Kaltenbach, and Lyons 1958). Cultures for smear preparation were centrifuged for 5 min at 700g. Cells were then washed in phosphate buffered saline; centrifugation and washing were repeated and the cells were resuspended in growth medium before a final centrifugation. Smears were prepared from the resultant cell pellet. Those for autoradiography were fixed in methanol but left unstained; the remander were fixed as above and stained in 10% Giemsa stain. Giemsa-stained smears were prepared from both labeled and unlabeled cultures, and the following parameters determined from cell counts and examinations: (a) Percentage of cells containing parasites. (b) Percentage of multinucleate cells. (c) Percentage of cells in mitosis (mitotic index).
caL
cumwm
65
(d) Percentage of cells in which the macroschizont stage of the parasite (normally present in parasitized bovine lymphoid cells grown in cul’ture) had transformed to the microschizont stage. Parameters (a)-(d) were determined from counts of 500 cells on each slide. (e) The mean number of nuclear particles (mean schizont number-MSN) in 50 intracellular macroschizonts per slide. The problems involved in such counts and the degree of accuracy expected have been previously discussed (Bamett, Brocklesby, and Vidler 1961) . Base values for parameters (a)-(e) were determined from a cell smear made prior to irradiation and culture division. (f) The size range of 100 intact, circular, mononucleate, parasitized cells. This was determined on examination of smears under a 100x oil immersion objective with 8x eyepieces, one of which contained a graticule marked into 100 equal divisions. Each cell was assigned to a size category based on graticule divisions (510, 10-15, 15-20, 20-25, 25-30, 30-35), and in this way a histogram of size range was constructed. Each graticule division represented 1.25 pm; actual size of cells could therefore be readily calculated. All smears were prepared and examined by the same person, and the same part of the smear examined on each slide. Autoradiography Smears for autoradiography were extracted in 5% aqueous trichloracetic acid for * hr at 4 C, and then processed as previously described (Irvin et al. 1974), using a 6-day exposure period. The following further parameters were determined from the examination of 500 cells per smear: (g) Percentage of unlabeled cells. (h) Percentage of cells with labeled nuclei.
66 (i) Percentage parasites.
IRVIN,
of
cells
with
BROWN
labeled
AND
STAGG
107 I
RESULTS
Viable Cell Count The division of cell cultures was calculated to yield a final cell concentration per 6 ml aliquot, of 5 x lo” cells per ml. However, following the various manipulations the number of viable cells fell to 3.6 x lo6 cells per ml; this was taken as the base value of cells in culture (Fig. 1). The figure also shows the changes in number of viable cells up to 4 days after irradiation. Nonirradiated cultures showed a regular increase in cell number up to Day 3, followed by a fall. Extrapolation based on Fig. 1 showed that nonirradiated culture had a mean doubling time of 28 hr. The nonirradiated culture that had [ sH] thymidine added showed a slight initial fall, but subsequently the growth rate paralleled that of the other nonirradiated culture at a lower level. Cultures irradiated at 300 rads showed a slight fall in number of viable cells up to Day 3, but then a marked rise with cell growth rate similar to that observed in nonirradiated cultures between Days 1 and 3. The [ 3H]thymidine labeled culture showed a similar growth pattern to the nonlabeled culture, but again at a lower level. Above 300 rads of irradiation no significant difference could be detected in viable cell counts between cultures with or without [3H] thymidine, the two values were therefore combined and the r.esultant mean plotted in Fig. 1. Cultures irradiated at 600, 900, and 1200 rads maintained a fairly constant viable cell number up to Day 2, then all fell sharply, the rate of fall being proportional to the amount of irradiation. Even at 1200 rads approximately lo4 cells were apparently viable after 4 days. Percentage of Cells Containing
Parasites
96.6% of cells in the preirradiation control smear were parasitized with macro-
1
2
4
3
Day.5
FIG. 1. Daily cell numbers/ml in Theileriu parua cultures irradiated at five different levels ( * = cultures labeled with [3H] thymidine).
schizonts of T. paroa. Subsequent smears showed a range from 95.6 to 99.470, but no significant differences as a result of different treatments, could be detected. Percentage of Multinucleate
Cells
6.2% of cells in the preirradiation control smear contained more than one nucleus. The range in the other smears was 0.8-10.470. In the nonirradiated cultures there was a slight fall, with time, in the percentage of multinucleate cells, and the lowest value was reached on Day 3. In the other cultures the highest values were usually found on Day 2, but no specific trend could be demonstrated, nor could any difference be detected between labeled and nonlabeled cultures. Percentage of Cells in Mitosis The mitotic index of the control smear was 2.870. The values for the other smears are shown in Fig. 2. No difference could be detected between labeled and unlabeled cultures so the values were combined and the means plotted in the figure. On Day 1 all cultures showed a similar value around 2%. The nonirradiated cultures maintained this value throughout the experimental pe-
IRRADIATION
EFFECI’S
ON
~hd!dU-HOST
riod, but all the others showed a marked rise up to a peak at Day 3, this rise being proportional to the amount of irradiation delivered. After Day 3 there was a sudden fall in the mitotic index in all cultures.
CELL
67
CULTURE
6or
Percentage of Cells Containing Microschizonts Microschizonts were detected intermittently in all cultures up to the end of Day 3, but never exceeded 0.4%. The value for the control smear was 0.270. Mean Schizont Nuclear Number (MSN) The MSN of the control smear was 13.4. The values for the other smears are shown in Fig. 3. In the unlabeled nonirradiated cultures there was a gradual fall in MSN after a slight initial rise. The labeled nonirradiated culture showed a similar pattern, but at a consistently higher level. The 300 rad group of cultures showed a pronounced initial rise and subsequent fall, and again the labeled cultures showed consistently higher values than the unlabeled ones. At 600 rads and above it was not possible to detect differences between labeled and unlabeled cultures, the two values were therefore combined and the means plotted in Fig. 3. The MSNs in the 600 rad cultures reached a peak on Day 2 and in the 900 and 1200 rad cultures reached peak on Day 3; following peak there was a rapid fall in MSN counts of all cultures. The peak 30 1
.c
4 1
2 Days
3
4
FIG. 2. Daily mitotic indices in Theileriu parua cultures irradiated in five different levels.
1
I 1
2
3
4
Days
FIG. 3. Daily values of mean schizont nuclear number in Theileria parua cultures irradiated at five different levels ( * = cultures labeled with L3H]thymidine).
values were in all cases proportional to the amount of irradiation delivered to the cultures. Cell Size The size range of cells is shown in Fig. 4. On Day 1 no significant difference was detectable, but on Day 2 there was an incr.easing percentage of large cells proportional to the amount of irradiation, this resulted in a shift of the histogram to the right at higher irradiation doses. This shift was most pronounced on Day 3, it was also generally greater in labeled than unlabeled cultures. The range of cell size is shown by the amount of horizontal spread of the histogram. This spread became more marked, with time, at higher irradiation levels, but in nonirradiated cultures the spread became less. Autoradiography The percentage of cells with labeled nuclei is shown in Fig. 5. The fall in per-
68
IRVIN,
BROWN
AND
% Controls
STACG
w 3HT Cultures
Rodiation
dose (rads)
..-
o-
10
I
20
30
0 10 Days parva
centage was proportional to the amount of irradiation to which cultures were exposed. Nonirradiated cultures maintained 100 r
80 . 70. 60 % 50 40. 30. 20 10
,
I
1
2 Days
3
30
3
2
FIG. 4. Size ranges of cells in Theileria (Each graticule division = 1.25 ,um).
t
20
600
3
FIG. 5. Percentage of cultured bovine lymphoid cells with [3H]thymidine incorporated into nuclei, following irradiation at five different levels.
cultures
irradiated
at five
different
levels.
a constant level until Day 3, but then fell. Irradiated cultures showed only a slight fall in percentage of labeled nuclei after 24 hr but then a rapid fall followed by recovery occurred. In the case of 600 and 900 rad cultures, the percentage reached a final level higher than that of nonirradiated cultures. The percentage of cells containing labeled parasites is shown in Fig. 6. In this case there was an increase in percentage proportional to the amount of irradiation to which cultures were exposed. No difference in [“Hlthymidine uptake between irradiated and nonirradiated cultures was detected until Day 2. The percentage of cells showing no label present, either in nucleus or parasite, was simply a reflection of the combined details recorded in Figs. 5 and 6, and no specific trends could be detected. The overall effect of irradiation on [“Hlthymidine uptake was that, with increasing doses of irradiation there was an increase in uptake
IRRADIATION
EFFECTS
ON
~hdf?h-HOST
CELL
CULTURE
69
of [3H]thymidine by the parasites but a decrease in uptake by the cell nuclei (Fig. 7). Parasite and Cell Morphology Figure 8 shows normal T. parva-infected bovine lymphoid cells, taken from the preirradiation control smear. In smears from cultures that had been irradiated the changes immediately noted were: the increase in cell size, the increase in mitotic index (with a majority of abrrormal mitoses), and the alteration in parasite morphology (Fig. 9). Macroschizont nuclear particles became smaller and increased in number, frequently to several hundred per schizont. In some cases cytomere-like blocks of schizont material were seen in infected cells (Fig. 10). Cultures examined 2-3 days after irradiation usually showed
%
FIG. 6. Percentage of cultured bovine lymphoid cells with [3H]thymidine incorporated into macroschizonts of Theileria paroa, following irradiation at five different levels.
FIG. 7. Autoradiograph of Theileria paruu-infected bovine lymphoid cells 2 days after irradiation at 900 rads. Culture was subjected to a 24-hr pulse of [‘Hlthymidine before harvesting. Counterstained with Giemsa X 1300. Three cells show normal nuclear labeling. In three other cells (arrowed) only the parasites are labeled; the largest of these cells is in mitosis and macroschizont nuclear particles are distributed among the nuclear segments of the cell.
70
IRVIN,
FIG. 8. Smear x1300.
of normal
Theileria
BROWN
AND
pawa-infected
two distinct populations of cells: those affected by irradiation undergoing the various morphological changes, and those of normal morphology, which were apparently unaffected by irradiation and were presumably destined to continue normal multiplication and repopulation of the culture. At higher doses of irradiation these appar.ently-normal cells were at a very low level, but even at 1200 rads some were continually present. DISCUSSION
The effects of irradiation on a cell culture system are very complex, and numerous physical factors relating to both the irradiation dose and the culture system have to be considered. Firstly the cells were nonsynchronized and the system examined here was further complicated by the presence of parasites in the cells, which themselves exert a profound influence on cell growth and behavior.
STAGG
bovine
lymphoid
cells.
Giemsa
stain
Cultures were examined for only 4 days, since beyond that time the accumulation of cell by-products and the exhaustion of growth metabolites would have provided further complicating factors in the interpretation of results. Even so, between Days 3 and 4, there was some evidence that these factors were becoming exerted, particularly in the nonirradiated cultures. Changing the medium could have circumvented these problems but, in itself, would have been a complicating factor. The addition of [ 3H]thymidine also altered the growth pattern of cells at 0 and 300 rads. At 600 rads and above, any effect of the addition of [ 3H]thymidine, was masked by the much greater effects of irradiation The nature of the [3H]thymidine effect was not examined, although its influence as an additional radiation source and as a DNA inhibitor could have both played some part (Evans 1966; Painter and Rasmussen 1964). Despite the complexity
IRRADIATION
EFFECXS
ON
%df&Z-HOST
CELL
CULTURE
71
FIG. 9. Smear of Theileriu parua-infected bovine lymphoid cells 3 days after irradiation at 1200 rads, showing irradiation giant cells, abnormal mitoses and increase in parasite mass. Giemsa stain X 1300.
of the system certain well-defined patterns emerged, and although the cell and parasite activities are closely integrated it is more convenient to consider separately their reactions to irradiation. Reaction of Cells to irradiation The general responses of mammalian cells to the effects of irradiation have been (Bond, reviewed by earlier workers Fliedner, and Archambeau 1965), whose descriptions have formed the basis of the interpretations of the current results. The initial handling procedures slightly depressed cell growth rate as shown by the nonirradiated cultures in the first 24 hr (Fig. 1). The initial fall in viable cell count of the irradiated cultures was probably partly due to handling but also as a result of death of cells in the very radiosensitive M and early S phases of the cell
cycle at the time of irradiation (Terasima and Tolmach 1963). Cells in the Gl and G2 phases could be expected to be less and consequently some radiosensitive would undergo normal division. This is shown by the slight rise in viable cell counts of irradiated cultures between Days 1 and 2. The fact that surviving cells were undergoing almost normal cell function for the first 24 hr, is further shown by the uptake of [ 3H] thymidine in their nuclei ( Fig. 5). This demonstrated that DNA synthesis was proceeding at a rate only slightly below that of nonirradiated cells. All cultures showed a similar slight fall in mitotic index at the end of the first day. No difference was noted between irradiated and nonirradiated cells since, although cells in mitosis would have been killed by irradiation, others would presumably have advanced along the cell cycle
72
IRVIN,
BROWN
FIG. 10. Smear of Theileria parua-infected at 1200 rads, showing two partially ruptured Giemsa stain X 1300.
AND
STAGG
bovine lymphoid cells 3 days after irradiation cells with cytomere-like blocks of macroschizonts.
to the M phase within the 24 hr following irradiation. Between Days 1 and 3 nonirradiated cultures were growing with an approximate doubling time of 28 hr ( Fig. 1) . During this time mitotic index and DNA synthesis remained more or less constant (Figs. 2, 5). After Day 3 however cells began to die, as shown by the falls in both viable cell count and uptake of [3H] thymidine by cell nuclei. This presumably occurred as a result of exhaustion of the cell medium. In the irradiated cultures the numbers of viable cells were still high at the end of Day 2 (Fig. 1) but many of these apparently viable cells were unable to synthesize DNA as shown by the rapid fall in percentage of labeled cells which started a day earlier (Fig. 5). The 300 rad culture had recovered from the effects of irradiation by the end of Day 3: DNA synthesis had returned to the normal level (Fig. 5) and, between Days 3 and 4, cell growth rate was similar to that observed in nonirradiated cultures between Days 1 and 3 (Fig. 1). The reason for this could have been that, in contrast to the nonirradiated cultures, the cell medium had not been exhausted by Day 3 (since earlier growth rate had been lower) and cells were able to multiply uninhibited.
Most cells which die following irradiation are capable of at least one division, and decreasing numbers can divide two, three or more times before death (Bond, Fliedner, and Archambeau 1965). This is demonstrated well (Fig. 1) for the viable cell counts of the 600, 900, and 1200 rad cultures; all fell steadily from Days 2 to 4 as increasing numbers of cells died in each culture. However, Fig. 5 shows that DNA synthesis was returning to normal levels on Day 3 in the 600 and 900 rad cultures, and on Day 4 in the 1200 rad cultures. In the 600 and 900 rad cultures the final levels recorded for percentage of labeled cells were, in fact, well above normal. This apparent revival of DNA synthesis may partly have represented cell recovery but also an accumulation of labeled cells which were unable to complete cell division. This is suggested by the rise in mitotic index proportional to irradiation (Fig. 2), indicating inability of cells to proceed beyond the M phase. In an earlier study Brown et al. (unpublished data) found that, although cells apparently survived irradiation above 600 rads, recovery of cultures only occurred at 600 rads and below. This was explained on the basis that cell numbers had fallen below the critical number of 4.3 x lo3 (Malmquist, Nyindo, and Brown 1970)
IRRADIATION
EFFECTS
ON
~heihia--HOST
and that toxic products had accumulated in the medium. There was an increasing incidence of radiation giant cells up to Day 3 proportional to the level of irradiation (Fig. 4). This presumably resulted from an increasing incidence of cells unable to divide but still able to grow in size. On Giemsa-stained smears, these giant cells were up to 4X the area of normal cells. Many giant cells were apparently unable to progress beyond the M phase, as shown by the increasing mitotic index related to irradiation dose and time. Most of the mitotic figures seen in giant cells appeared to be abnormal metaphases, and the mechanism responsible for further progression was, in many cases, apparently deficient. After Day 3 there was a general fall in the percentage of giant cells (Fig. 4) presumably due to cell death and lysis. This explains the fall in mitotic indices by Day 4 (Fig. 4), and further emphasizes the gen.eral recovery of cell cultures at this time. Reaction of Intracellular to Irradiation
Parasites
Under normal circumstances macroschizont and host cell activities are apparently synchronized, since the MSN of the macroschizonts does not appreciably alter with successive passages of culture (Hulliger, Brown, and Wilde 1966). Furthermore the fact that neither free macroschizonts nor nonparasitized cell can apparently grow on their own, suggests that each is dependent on the other. This interdependence however, is disrupted by irradiation, and parasite and host cell respond in different ways, the cell being more radiosensitive than the parasite. Purnell et al. (1974) have shown, for example, that T. parua in the tick, is unaffected by irradiation doses up to 10 krad and a decreasing proportion of parasites can survive doses up to 45 krad. Cell division was increasingly inhibited at higher irradiation doses. Parasite division appeared however to be unaffected
CELL
CULTURE
73
and in arrested cells the MSN sometimes became very high. The fact that the mean MSN was highest in the 1200 rad cultures (Fig. 3) may simply be a reflection of the fact that these cultures contained the highest percentage of arrested cells. Other agents or factors which arrest or retard cell division, such as colchicine (Hulliger 1965; Hulliger et al. 1964), actinomycin D (Moulton, Krauss, and Malmquist 1971) and raised incubation temperature (Hulliger, Brown, and Wilde 1966), have been shown to result in similar MSN increases, Two other possibilities must however be considered in relation to increased MSN following irradiation: firstly the parasite may have been directly stimulated by irradiation to undergo division; the increasing percentage of labeled parasites with higher doses of irradiation, and the observed morphological changes in the parasite suggested that there was increased and altered activity in the macroschizonts. Other work has shown, for example, that T. parva macroschizonts do not readily incorporate [ 3H] thymidine under normal conditions (Irvin et al. 1974; Moulton, Krauss, and Malmquist 1971). Secondly, the altered environment of the cell may have affected parasite behavior, since rapid nuclear division of macroschizonts and the formation of cytomere-like blocks (Fig. 10) have also been observed in bovine hamster heterokaryons parasitized with T. parva (Irvin et al. 1975). A comparison of the uptake of [ 3H]thymidine in colchicine-arrested cells (where the parasite is less likely to be directly affected by the cell inhibitor) with [3H]thymidine uptake in irradiated cells, might help to evaluate these questions. So far it has not been possible to irradiate free macroschizonts to examine the direct effects of irradiation on this stage of the parasite. There was no evidence in this work that irradiation speeded-up the switch of the parasite from macroschizont to microschizont, and, although many of the later
74
IRVIN, BROWN AND STAGG
schizonts contained large numbers of small particles, they morphologically resembled macroschizonts rather than microschizonts. However, Brown et al. (unpublished data) have shown that different passage levels ‘of culture show different propensities for microschizont formation, and in one .experiment these workers showed that irradiation did result in an increased incidence of microschizont formation. It has also been shown in other systems where cell division has been r.etarded that an increased percentage of microschizonts occurs (Hulliger, Brown, and Wilde 1966). Many factors need further investigation and, most important of all, the infectivity and possible attenuation of the parasites need to be evaluated by in viva studies. ACKNOWLEDGMENTS We are grateful to M. P. Cunningham and our colleagues on the Project for help and advice. Our thanks are also due to Professor G. Mugera for allowing us access to the O°Co source at the Faculty of Veterinary Science, University of Nairobi, and to R. C. Payne and Mrs. S. Stagg for assistance with photography and diagrams. The technical assistance of L. Njuguna is gratefully acknowledged. This paper is published by the kind permission of Dr. G. L. Corry, The Director, EAVRO. The Project is supported by the United Nations Development Programme, with the Food and Agriculture Organization of the United Nations as the Executing Agency, in cooperation with the East African Community. The Project is also supported by the Overseas Development Administration of the United Kingdom (Research Project R2845), the United States Department of Agriculture, the Rockefeller Foundati’on, the Intemational Atomic Energy Agency, and the Pfizer Corporation.
REFERENCES BARNETT, S. F., BROCKLESUY, D. W., AND VIDLER, B. 0. 1961. Studies on macroschizonts of Theileria parva. Research in Veterinary Science 2, 11-18. BOND, V. P., FLIEDNER, T. M., AND ARCHAMBEAU, J. 0. 1965. “Mammalian Radiation Lethality.” Academic Press, London. CUNNINGHAM, M. P., BROWN, C. G. D., BURRIDGE, M. J., MUSOKE, A. J., PURNELL, R. E., AND
DARGIE, J. D. 1973. East Coast fever of cattle : “Co irradiation of infective particles of 20, Theileria partia. Journal of Protozoology 298-300. EVANS, E. A. 1966. “Tritium and its Compounds.” Butterworths, London. HULLIGER, L. 1965. Cultivation of three species of Theileria in lymphoid cells in uitro. Journal of Protozoology 12, 649-655. HULLIGEH, L., BROWN, C. G. D., AXD WILDE, J. K. H. 1966. Transition of developmental stages of Theileria parva in vitro at high temperature. Nature (London) 211, 328-329. HULLIGER, L., WILDE, 1. K. H., BROWN, C. G. D., AND TURNER, L. 1964. Mode of multiplication of Theileria in cultures of bovine lymphocytic cells. Nature (London) 203, 728-730. IRVIN, A. D., BHOWN, C. G. D., BOARER, C. D. H., CRAWFORD, J. G., AND KANHAI, G. K. 1974. Autoradiographic evidence for the occurrrence of cell fusion in cultures of Theileria-infected bovine lymphoid cells. Research in Veterinary Science 16, 137-142. I~IVIN, A. D., BROWN, C. G. D., STAGG, D. A., KANHAI, G. K., AND ROWE, L. W. 197.5. Hybrid cells, infected with Theileria parua, formed by fusion of hamster and mouse cells with parasitized bovine lymphoid cells. Research in Veterinary Science, in press. KALTEXBACH, J. P., KALTENBACH, M. H., AND LYONS, W. B. 1958. Nigrosin as a dye for differentiating live and dead ascites cells. Experimental Cell Research 15, 112-117. MALMQUIST, W. A., NYINCO, M. B. A., AND BROWN, C. G. D. 1970. East Coast fever: Cultivation in zjitro of bovine spleen cells infected and transformed by Theileria parva. Tropical Animal Health and Production 2, 139-145. MOULTON, J. E., KRAUSS, H. H., AND MALMQUIST, W. A. 1971. Growth characteristics of Theileria puma-infected bovine lymphoblast cultures. American Journal of Veterinary Research 32, 1365-1370. PAINTER, R. B., AND RASMUSSEN, R. E. 1964. A pitfall of low specific activity radioactive thymidine. Nature (London) 201, 409-410. PURNELL, R. E., BROWN, C. G. D., BURRIDGE, M. J., CUNNINGHAM, M. P., EMU, H., IRVIN, A. D., LEDGER, M. A., NJUGUNA, L. M., PAYNE, R. C., AND RAULEY, D. E. 1974. ‘“Co irradiation of Theileria parua in its tick vector Rhipicephalus appendiculatus. International Journal for Parasitology 4, 504-511. TERASIMA, T. T., AND TOLMACH, L. J. 1963. Variation in several responses of Hela cells to X-irradiation during the division cycle. Biophysics Journal 3, 11-33.