Growth of protozoa in tissue culture. II.—Plasmodium relictum, exoerythrocytic forms

183 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.' Vol. 40. No. 2 . October, 1946.

GROWTH OF PROTOZOA IN T I S S U E CULTURE.

H.--PLASMOD1Ubi RELICTUM, EXOERYTHROCYTIC FORMS. BY FRANK HAWKING.*

From the National Institute for Medical Research, London, N.W. 8.

This paper describes the growth of the exoerythr0cytic forms of Plasmodium relictum in tissue culture by the technique already described for P. gallinaceum (HAWKINC, 1945). TECHNIQUE. Canaries were inoculated intravenously each with the glands of about eight mosquitoes (C. pipiens) infected with the G strain of P. relictum. T h e y were killed about the 8th day when trophozoites were beginning to appear in the erythroeytes. At this time, smears made from the spleen and liver contained only few exoerythrocytic forms (none to four in fifty fields). Small pieces of spleen were set up for tissue culture by the technique already described (HAwKINO, 1945). T h e fluid medium in the early experiments contained 10 to 20 per cent. of canary serum, but such serum was difficult to obtain and necessitated the sacrifice of many canaries. In later experiments i t was found that the canary cells grew fairly well when they were embedded in a small amount of fowl plasma and were bathed in a fluid consisting of 20 per cent. fowl serum, 20 per cent. chick embryo extract, 60 per cent. Tyrode plus penicillin to about 3 units per c.c. and enough phenol red to give a pink colour. T h e medium was changed about every 5 days. Growth of the canary cells in this medium was not quite as satisfactory as that of ordinary chicken tissue ; the tissue tended to form an annular mass of elongated cells around a clear open space containing only the necrotic remains of flattened cells. Consequently the parasites were partly hidden by the thick cell mass in which they occurred, and they were less easy to study than was P. gallinaceum. For microscopical examination, one or more of the coverslips carrying the cultures was removed and fixed in Schaudinn's fixative. It was stained in Giemsa, differentiated in very dilute acetic acid for a few seconds, dehydrated by passing through appropriate mixtures of acetone and xylol, and mounted in a neutral mountant. In many of the tissue cultures which have been made during this period, trouble was experienced due to the presence of small slender bacilli, growing in groups, often in the macrophages or in masses of material like mucoid. T h e y did not appear until the 4th to 6th day, and they impaired the vitality of the culture without destroying it altogether. T h e y could be removed by passing the embryo extract through a filter; so apparently they came from the chick embryos. Filtration through a Seitz filter impaired the growthpromoting properties of the extract, but filtration through a collodion one was less deleterious. T h e growth of these bacilli was not prevented by penicillin or by streptomycin about 5 units per c.c. * Grateful acknowledgments are due to Miss A. BISHOP, D.SC., for valuable help; to Miss V. D. MARKHAM,Miss R. J. BERSON, and Miss V. PICKERTONfor assistance with the experiments and the illustrations; to Mr. F. V. WELSH, F.R.M.S., and Mr. C. D. SUTTON for the photography, and to Dr. S. A. WAKSMAN for the supply of streptomycin.

184

P. R E L I C T U M , EXOERYTHROCYTIC FORMS

EXPERIMENTAL RESULTS.

Successful cultures were made from the spleens of three canaries, and they were maintained up to 19 days, after which all the remaining colonies were removed for microscopical examination. Parasites were found both at the earliest time of examination (4th day) and at the latest (19th day). Fluid was removed from some of the flasks on the 5th and 9th days of cultivation and was injected into a canary which became infected 14 days after the first inoculation. I n some of the cultures very vigorous growth of the parasites occurred, but as with P. gallinaceum it was unevenly distributed. In some places parasites were very numerous, in other adjacent areas they were completely absent. A p p a r ently the merozoites do not spread very far from the spot where they have been liberated. G o o d growth of the parasites occurred in the presence of streptomycin, 5 units per c.c. W h e n small pieces of liver were implanted in the flasks instead of spleen, there was little or no growth of cells. MORPHOLOGY OF P.

relictum

IN TISSUE CULTURE.

M a n y different forms of the plasmodium were seen in the cultures, and b y selection of suitable types, the probable cycle of development can be illustrated. But since a single parasite cannot be followed t h r o u g h o u t the whole course of the cycle, one cannot be certain whether some of the forms seen are really stages through which all parasites pass, or whether they are only abnormal appearances which stand outside the regular cycle. It is probably safest to describe the main types, recognizing that the divisions between t h e m are arbitrary, and to realize that the relation of o n e type to another is assumption rather than observed fact. Accordingly, the following stages may be recognized. 1. Small forms with one piece of chromatin (Figs. 1 and 1 4 ) . - - T h e s e are usually r o u n d e d and measure about 2"5/z across. T h e chromatin is round or oval and measures 1 to 1.6/, ; it stains pink or crimson with Giemsa. Sometimes the outer parts stain more clearly than the centre, sometimes the staining is uniform. T h e cytoplasm stains pale or dark blue with Giemsa ; sometimes there are one or two small vacuoles in it. Often there is a small darkly staining FIGS. 1 TO 3.--Parasites with one to four pieces of chromatin. FIG. 4.--Parasite with five pieces of chromatin ; cytoplasm rather granular. FIG. 5.--Parasite with seven pieces of chromatin; cytoplasm is granular and contains

vacuoles. Fro. 6.--Schizont with many pieces of chromatin (many at other optical levels, not shown). The cytoplasm is beginning to be concentrated round some of the pieces of chromatin. FIG. 7.--Schizont forming merozoites. FIG. 8.--A mature schizont containing many merozoites arranged irregularly (others at different optical levels, not drawn); some merozoites are emerging into the adjacent: tissues. FI~, 9.--A schizont breaking up into merozoites. ( × 1,900; Drawn by V. D. M.). FIG. 10.--A schizont at a later stage of disintegration (see text). FIG. l l . - - A group of free merozoites. (Drawn by F. H.) Magnification according to scale (except Fig. 9).

FRANK H A W K I N G

185

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6

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8

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126

P. R E L I C T U M , EXO]~RYTHROCYTIE FORMS

granule in the cytoplasm near the chromatin. (All recorded dimensions depend greatly on the degree of flattening of the parasite ; flattened parasites naturally appear much larger under the microscope than those of equal volume but spherical shape. Since flattened parasites are easier to study and photograph than spherical ones, most of the illustrations and measurements are made on flattened ones.) 2. Large forms with one piece of chromatin (Fig. 15).--These are irregular in size and shape but often measure 3.5/, across. The chromatin is usually elongated (1.6 x 0-8/~) but sometimes it is rounded. Often there is a clear space between the chromatin and the part of the cytoplasm which stains bright blue with Giemsa. A granule and vacuoles may be present. Presumably these forms represent the further growth of (1). 3. Forms with several pieces of chromatin (up to twenty or more), as illustrated (Figs. 2 to 5).--In the smaller forms the cytoplasm is abundant and stains bright blue, Granules and vacuoles may be present. Presumably these forms arise from the division of the chromatin shown in Fig. 2. 4. LaNe schizonts with twenty or more pieces of chromatin (Figs. 6, 12, 16). - - T h e s e may be rounded or oval and may measure 9 to 14tL according to the volume and the degree of flattening. The number of pieces of chromatin ranges up to forty-five or more, the larger numbers being difficult to count accurately. These schizonts may be divided into two types. (a) With chromatin staining crimson with Giemsa and a fair amount of cytoplasm (staining bright blue) distributed approximately evenly between the pieces of chromatin ; presumably these are a development of (3). (b) With chromatin staining more deeply and scanty cytoplasm which is concentrated closely round the piece of chromatin so that the intervening spaces appear empty. (Fig. 6.) 5. Forms in which merozoites are foreshadowed.--These forms are as big as those of (4). The pieces of chromatin stain dark crimson and each is surrounded with a little dense cytoplasm which may be rounded, or may be elongated as in a mature merozoite. These early merozoites may be arranged in three ways: (a) packed together apparently irregularly with many of the outer pieces lying tangentially. Some of these look as though the schizont had been converted into a cluster of merozoites arranged at random, which were gradually falling apart (Fig. 8) ; (b) with two clear areas, surrounded by early merozoites, some spherical, others elongated and lying radially. Some have twenty four merozoites, some more (Figs. 7,13) ; (c) elongated merozoites arranged radially round a small dark mass in the centre which might be ckromatin or cytoplasm (difficult to distinguish) as in Fig. 9. This form has about twenty-one pieces of ckromatin. Only one or two forms like this were seen. The form shown in Fig. 10 is interesting. There are several merozoites lying free and several others arranged radially round a central mass which stained dark purple with Giemsa. Photographs revealed that this dark central mass had the structure shown in the drawing; there are eight or more pieces

FIG.

EL--Large

schizont.

FIG. 13.-Three

The

schizonts

longitudinal Cultivated

changing

clear space is presumably 18 days.

into merozoites.

Plasmodium relictum

To face jmge 186.

x 1,250.

Cultivated

an artefact

12 days.

FIG.

14

FIG.

15

FIG.

1.6

FIG.

17

Plasmodium FIG. 14.-Two FIG. I5.-Many

FIG.

17.-A

small forms larger forms

FIG. 16.-A group of

relicturn

x

1,250.

with a single piece of chromatin. Cultivated 19 days. with single pieces of chromatin ; other forms in adjacent cells. Cultivated 12 days. large schizont and a smaller one. Cultivated 12 days. merozoites in a large flattened cell. Cultivated 12 days.

FRANK HAWKING

187

of chromatin, some of which are partly differentiated into merozoites arranged radially. The appearances suggest merozoites being liberated in two successive crops from a central core, as suggested by HuFF and COULSTON (1945) for the meta-cryptozoites of P. gallinaceum. No other form was found in which this appearance was detected, but this failure may be due to t h e fact that most other forms, at this stage of schizogony, were overlaid by cells. It is not clear whether (a), (b) and (c) are successive or alternative stages in the development of the parasite. In particular, it is difficult to reconcile form (a), in which the fully developed merozoites are arranged at random, with a cycle involving forms (b) and (c) in which the merozoites are arranged radially. 6. Free merozoites (Figs. 11, 17).--These are elongated forms about 0"8~ wide by 2 to 2.5~ long. One end is pointed and the other is often rounded, or square. The chromatin is spherical, about 0-8/z across. Some authors have described macro-merozoites and micro-merozoites in the development of the exoerythrocytic forms of P. relictum. Only one type (as above) has been seen in these cultures. DISCUSSION.

The main purpose of this investigation was to demonstrate that the tissue culture technique, which had been developed for the study of P. gallinaceum, was applicable also to P. relictum. It was pointed out in the previous paper (HAWKING, 1945) that any intracellular parasite, which lives inside a type of ceil which can be grown in vitro, ought to prove susceptible to cultivation in this manner. The parasites which have now been grown in this way include P. gaUinaceum ; P. relictum and P. lophurae (ToNKIN and HAWKING, in press), Trypanosoma cruzi (HAWKING, in press) and Leishmania donovani (HAWKING, in preparation for publication). In addition the growth of P. cathemerhtm in a single experiment was reported by HEGNER and WOLFSON (1939); and TCHERNOMORETZ (1945) has reported the cultivation of Theileria annulata by a technique which he had devised independently but which is similar to the one employed here. Cultivation of parasites which grow in small animals (e.g., Leishmania in hamsters) or small birds (e.g., canaries) is much facilitated by the use of heterologous sera obtained from larger animals. In the present instance fowl serum was not completely satisfactory for the growth of the cells and perhaps the serum of some other species would have been better. This technique is advantageous for studying the morphology of the exoerythrocytic forms, especially in the stages leading up to schizogony and the formation of merozoites. No clear description of these has been discovered in the previous papers on the exoerythrocytic forms of P. relictum (R~FAELE, 1936 ; HEGNER and WOLFSON, 1938 ; MANWELL, 1940 ; REICHENOW and MUDROW, 1943), although some illustrations contain forms suggestive of

188

P. R E L I C T U M ,

EXOERYTHROCYTIC FORMS

elongated]merozoit~s. As described above, the merozoites of P. relictum assume their elongated form at an early stage of schizogony, but the exact relationship of some of the forms seen, one to another, is not clear. In the previous work on P. gallinaceum, a simpler method of fixing and mounting the preparations was u s e d ; therefore, an attempt to compare the morphologies of the two plasmodia in tissue culture is not desirable in this place. REICHENOW and MUBROW (1943) described two types of merozoite ( m a c r o - and micromerozoites) being formed during the pre-erythrocytic stages of P. relictum and HUFF and COULSTON (1945) noted two similar types in the pre-erythrocytic development of P. gallinaceum. In these cultures only one type of merozoite, corresponding presumably to macro-merozoites, was seen. SUMMARY.

The exoerythrocytic forms of Plasmodium relictum have been grown in tissue culture, using the technique previously described for P. gallinaceum. Growth was terminated after 19 days. Fluid removed from the culture flasks after 5 and 9 days was infective for a canary. Growth took place in the presence of penicillin and of streptomycin. This technique is advantageous for studying the morphology of the exoerythrocytic forms, especially in the different phases of schizogony. T h e various stages are illustrated. REFERENCES. HAWKING,F. (1945). Growth of protozoa in tissue culture. I. Plasmodium gallinaceum, exoerythrocytic forms. Trans. R. Soc. trop. Med. Hyg., 89, 243. (1946). Growth of protozoa in tissue culture. II. Plasmodium relictum. Ibid., 40; 183. Growth of protozoa in tissue culture. III. Trypanosoma cruzi. Ibid. In press. HEGNER, R. • WOLFSON, F. (1938). Toxoplasma-like parasites in canaries infected with Plasmodium. Amer. ft. Hyg., 27, 212., (1939). Tissue culture studies of parasites in reticulo-endothelial cells in birds infected with Plasmodium. Ibld., Sect. C., 29, 83. HUFF, C. G. & COULSTON,F. (1945). The development ofPlasmodium gallinaceum from sporozoite to erythrocytic trophozoite, ft. inf. Dis., 75, 231. MANWELL, R. D. (1940). Life-cycle of Plasmodium relictum var. matutinum. Amer. ft. trop. Med., 20, 859. RAFFAELE, G. (1936). Presumibili forme iniziali di evoluzione di Plasmodium relictum. Riv. Malariol., Anno 15 (11 Nuova Serie). Sezione 1, 318. REIeHENOW, E. & MUDROW,L. (1943). Der Entwicklungsgang von Plasrnodium praecox im VogelkSrper. Dtsch. Tropenmed. Z., 47, 289. TCHERNOMORETZ, T. (1945). Multiplication in vitro of Koch bodies of Theileria annulata. Nature, 156, 391. TONKIN, I. M. & HAWKINC,F. Growth of protozoa in tissue culture. IV. Plasmodium • lophurae. Trans. R. Noc. trop. Med. Hyg. In press.