The life cycle of the coccidian parasite, Toxoplasma gondii, in the domestic cat

380 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 65. No. 3. 1971.

SPECIAL ARTICLE THE LIFE CYCLE OF THE C o c c i D I A N PARASITE, TOXOPLASMA GONDII, I N THE DOMESTIC CAT W. M. H U T C H I S O N AND J. F. D U N A C H I E

Department of Biology, University of Strathclyde, Glasgow, C. 1, Scotland AND

K. W O R K AND J. CHR. S I I M

Department of Toxoplasmosis and Viral Diseases, Statens Seruminstitut, Copenhagen, Denmark In previous years, research activity on Toxoplasma gondii has been concentrated on the pseudocysts or terminal colonies (containing "trophozoites') and the tissue cysts (containing zoites). The "trophozoite" is a proliferative stage of Toxoplasma which seems to be capable of infecting and multiplying within many nucleated cells.* Cells parasitized subsequently rupture and the "trophozoites" escape to continue their intracellular multiplication elsewhere. T h e tissue cyst wall consists of a resilient membrane which encloses the zoites; these are most frequently observed embedded in the C.N.S. and in the musculature. Since the pseudocysts and the tissue cysts were the only two forms of Toxoplasma which were known, the earlier workers were forced to attempt to explain the transmission and the life cycle in terms of these. Such explanations were facilitated by the fact that both forms were transmissible under natural conditions. However, for reasons reviewed by HUTCmSO~ et al. (1969b), it became apparent that unknown stages of Toxoplasma existe& WORK and HUTCHISON (1969a, b) ultimately isolated coccidianlike cysts from the faeces of cats which had previously been fed with the tissue cysts of Toxoplasma from mouse brains. These faecal cysts were capable of inducing Toxoplasma infections in mice to which they were fed and had a resistance to environmental influences far in excess of that of the pseudocysts and the tissue cysts. It was difficult to avoid the conclusion that these faecal cysts were Isospora-like coccidian o6cysts not only on the It is a pleasure to acknowledge the advice and encouragement of Professor P. C. C. Gamham. Dr. C. A. Hoare gave us valuable advice on taxonomy. We are also grateful to the following for help in this work; Drs. E. Canning, L. P. Joyner, D. K. Blackmore, M. Festing, R. Killick-Kendrick; Sir William and Lady Weipers; Misses E. M~ller and D. G. Owen; Messrs. C. Norton, R. F. Parrott and J. Eveleigh. For technical assistance we are indebted to Mr. D. Dougherty and Miss M. Reilly. We are indebted to the M.R.C., L.A.C. for the SPF cats. Work at Strathclyde University is supported in part by grants from the Medical Research Council, the Wellcome Trust and the World Health Organization. Work at the Department of Toxoplasmosis and Viral Diseases, Statens Seruminstitut, Copenhagen, is aided in part by grants from the King Christian X Foundation, and the U.S. Public Health Service, Research Grant Division (Ai 01741), Bethesda, Md., the World Health Organization, and Cold Stores Foundation. ~We are using the term "trophozoite" within quotation marks when referring to it in this toxoplasmic sense. We use it without quotation marks, in its correct sense, to denote the growing, pre-schizogonic stage in the intestinal epithelium of the cat.

W. M. HUTCHISON, ]. F. DUNACHIE, K. WORK AND J. CHR. SIIM

38i

basis of their morphology b u t also because they underwent a sporulation process identical to that observed in the coccidian genus Isospora. However, at this stage, evidence of schizogony and gametogony in the epithelial cells of the intestine or of associated organs was lacking. Consequently, the faecal cyst was tentatively referred to as the "new cyst" rather than as an ogcyst. T h e required evidence of schizogony and gametogony was obtained by HUTCHISON et al. (196%, 1970) and FRENr:EL et al. (1970). It had also been shown (SLIM et al., 1969) that the new cyst was a disporocystic and tetrazoic structure and this gave supporting evidence that Toxoplasma was a coccidian parasite and that the cyst, which WORK and HUTCHISON (1969a, b) had observed, was indeed an o6cyst. Simultaneously with these observations, SHEFFIELD and MELTON (1970) also came to the conclusion that the infective form of Toxoplasma found in cat faeces was an o6cyst resembling those of the genus Isospora. T h e purpose of the present paper is to describe and discuss in greater detail the stages which we have observed in the intestinal epithelium of the cat.

Materials and methods T h e Strain A mice used in these experiments were offspring of a breeding nucleus provided by the Imperial Cancer Research Fund, London, in 1954. No natural infection with Toxoplasma has ever been observed in the tissues of several thousand of these mice which have been examined as uninfected controls. The strains of mice at the Statens Seruminstitut were Toxoplasma-negative, being examined both serologically and parasitologically. The cats were male and were obtained from the Specific Pathogen Free (SPF) cat colony of the Medical Research Council's Laboratory Animals Centre. I n addition to being free of all known feline viral pathogens, these cats were guaranteed to be free of both coccidian infectious and those caused by Toxoplasma gondii. We have verified these guarantees by preliminary faecaland serological examination of the cats which were used in our experiments. The breeding queens in the colony were also dye-tested by us for Toxoplasma antibodies; all were negative. The SPF cats which we used were sent to us by air in large polystyrene boxes which had previously been sterilized; the lid formed an airtight seal. The inmates obtained air through bacterial filters set in the side of the box; this ensured that they were not exposed to infection en route. At Glasgow, they were maintained in sterilized accommodation and fed on sterilized milk and sterile cat food. I n this situation there was no possibility of acquiring either toxoplasmic or coccidian infections, as was evidenced by the absence of both infections in control animals at autopsy. The suitability of SPF cats for these experiments wiU be raised again in the discussion to this paper.

T o x o p l a s m a strains Two strains of Toxoplasma were used in our experiments. Firstly, the Beverley strain was used in the majority of the experiments and is maintained at Strathclyde University by the subcutaneous injection of approximately 50 tissue cysts into each mouse. These cysts were obtained from the brains of previously infected mice. Secondly, Statens Seruminstitut strain 119 was used to infect Cat SPF 8. This strain is maintained by intraperitoneal passage of infected mouse brain in Copenhagen; it was originally isolated from a pig. As the serological aspects of this work were carried out in Copenhagen, it was more convenient to use strain 119 for a controlled experiment which would eliminate the remote possibility that unknown stages of cat coccidians were present in the mouse brains which we were feeding to the SPF cats. I n order to show that such stages could not be responsible for the o6cysts in the cat faeces, 2 groups, each of 30 mice, were taken. One group was bled and dye-tested. The other group, which was also pre-bled, was infected intraperitoneally with the tissue cysts of strain 119. After 30 days, both groups were bled and dye-tested; the mice were then killed and the brains removed and sent from Copenhagen to Glasgow. At Strathclyde, both the positive and negative batches of mouse brains were checked microscopically. The batch containing the tissue cysts was then emulsified and an aliquot was inoculated subcutaneously into each of 12 mice; a similar procedure was adopted with the negative brains. The positive emulsion, containing strain 119, and the negative emulsion were fed to SPF 8 and SPF 9 respectively, as described in a following section.

382

L I F E CYCLE O F T H E C O C C I D I A N P A R A S I T E , T O X O P L A S M A

G O N D I I I N T H E D O M E S T I C CAT

T h e use of strain 119 also enabled us to compare its infectivity with that of the Beverley strain.

Cats fed with tissue cysts (a)

Beverley strain 9 of the cats indicated in Table I were fed with aseptic mouse brains containing the tissue cysts of the Beverley strain of Toxoplasma.T h e mouse carcasses were discarded. Th e faeces of all cats were examined prior to this meal. After being fed, the faeces were recovered at daily intervals. T h e ZnSO4 separations were performed on all the stools, as described by HUTCHISON (1967) and a number of separations were obtained. T h e faeces of uninfected control cats were processed in an identical manner. All separations were examined microscopically for Toxoplasmao6cysts and were stored in 2"5~o potassium dichromate. After 14 days storage at room temperature, aliquots from. each separation were each fed to a group of 4 mice regardless of the presence or absence of o6cysts. 4 mice were set aside as controls. This procedure was followed with all separations. After 35 days, the brains of all mice were examined for tissue cysts and the sera were dye-tested~ Positive or negative results were then related to the presence or absence of o6cysts in the cat. (b)

Statens Seruminstitut strain 119 Cats S P F 8 and S P F 9 were dye-tested immediately prior to our experiments with them and also 30 days in advance of this. T h e two dye-tests at 30 day intervals both on the cats and on the mice whose brains were fed to Cat SPF 9, provided a double check on the negativity of these animals. T h e emulsion containing the tissue cysts of strain 119 was fed to Cat S P F 8 and the negative emulsion to Cat SPF 9. T h e faecal production of both cats was dealt with as in the previous section.

Cats fed with o6cysts Cats S P F 4, 13, 14, 15 and 17 were fed with the o6cysts of Toxoplasmarecovered from the tissue cyst-ifiduced infections of earlier S P F cats. Their faeces were processed as previously described.

Serology All sera were coded and examined by routine dye-test procedures used at the Statens Seruminstitut and staridardized by AAGAARD(1960). Titres of less than 1 : 5 are considered to be negative.

Cat autopsy Th e cats were killed by sodium pentothal injection and the blood was immediately removed for dye-testing. Apart from Cats S P F 2, 3 and 5 which have been described separately (HUTCHISON et al., 1970), the entire small intestine of each of the remaining cats indicated in Table I was removed and divided into 24 equal parts. Portions, approximately 1 cm. in length, were removed from each part and fixed for the histological examinations described below. T h e remainder of each part was used to make impression smears.

Histological procedures Impression smears were fixed in Bouin's fluid and stained with Giemsa. From these smears, the numbers of merozoites in mature schizonts were counted. Most material prepared for sectioning was fixed in Camoy's fluid (2 changes of 3 hours) and taken up through 90% (2 changes) and 95% ethanol to equal parts of absolute ethanol and ester wax, with 2 changes of ester wax before embedding. Some material was also fixed in Zenker's fluid and in 10% neutral formol saline. T h e optimum thickness for sections appears to be 4~z. T h e Giemsa/Colophonium method of BRAY and GAgNHAM (1962) gave the best staining results. Unfortunately, this technique cannot be adapted for the simultaneous processing of a number of slides because, after removal from their Giemsa solution, each slide must be individually differentiated and dehydrated precisely in the manner described by these authors. After excess stain has been washed off the slide with water and the undersurface dried, the most critical stage of the operation foUows, i.e. differentiation. This involves fl6oding the hand-held slide with the colophonium-acetone solution. While this is being added (preferably with a wash-bottle and over a sink), the slide is tilted back and forth to

W. M. HUTCHISON , J. F. DUNACHIE, K. WORK AND J. CHR. SIIM

383

obtain a colophonium-acetone-water mixture. Good differentiation depends on the amount of water which is retained on the slide and mixes with the colophonium-acetone, and great care must be taken to avoid losing the mixture over the edge of the slide. More of the reagent must be added as the acetone' evaporates and differentiation slows down as the proportion of colophonium in the mixture increases. The slide is then dehydrated rapidly by flooding with acetone/xylene mixtures (2 : 1, 1 : 2) and passed into xylene; it should be drained, but not dry before mounting with euparal. The nuclear detail of the organism is best seen with Feulgen, methyl green-pyronin, or Heidenhain's iron haematoxylin. The parasites, stained with Ehrlich's haematoxylin and eosin, do not contrast as well with the host tissues. However, PAS, followed by Ehrlich's haematoxylin is a good combination for the demonstration of polysaccharides in the parasites, particularly in the gametocytes.

Results The ages of the breeding queens in the Medical Research Council's SPF cat colony ranged from 9 months to 3½ years; their weights varied from 2.68 to 4-3 kg. M1 were seronegative, having Toxoplasmadye-test titres of less than 1 : 5. The outcome of the experiments with the male offspring of these female cats is summarized in Table I. M1 these were bled prior to our experiments with them and were sero-negative ( < 1 : 5) for Toxoplasma; the faeces examined during this period were also negative for Coccidia. The weights of Cats SPF 1-7 were not recorded. In this paper we are mainly concerned with an investigation of schizogony and gametogony and have examined most cats during the early phase of o6cyst production when all these stages of development can be observed simultaneously. As can be seen, we have concentrated on infections using the tissue cysts of Toxoplasma. Our work on infections using o6cysts is of a preliminary nature. Cats fed with tissue cysts (a) Beverley strain SPF 1 was killed 3 days after being fed with tissue cysts from mouse brains; at this time the cat was still sero-negative. No o6cysts had been seen in the faeces and no developmental stages were seen in the epithelium of the small intestine. A similar state of affairs was observed in SPF 10, which was killed after 4 days. Cats SPF 2, 3 and 5 established that Toxoplasmawas a coccidian parasite undergoing typical schizogony and gametogony in the intestinal epithelium of the cat and that these processes lead to the appearance of o6cysts in the faeces. This experiment has already been described by HIJTCHISONet al. (1969a, 1970) and needs no further comment except that no sero-conversions were observed. Neither was a conversion observed in another experimental cat (SPF 12) autopsied on the 5th day, despite the fact that all stages of development were present in the intestinal epithelium. In Cat SPF 16, a low positive titre (1 : 10) was observed on the 6th day; again all stages of schizogony and gametogony were seen. Cats SPF 6 and 7 were used solely for the production of o6cysts for experimental purposes and consequently their intestines were not examined histologically. O/Scysts were first shed on the 10th day after feeding tissue cysts to both these cats. They were killed 9 days later; both animals had become sero-positive with a dye-test titre of 1 : 250. M1 other cats fed with the Beverley strain were used principally to provide material for histological examination (see Table I). (b) StatellS Seruminstitut strain 119 The outcome of the experiment in which Cat SPF 8 was fed with the brains of mice containing cysts of Toxoplasma strain 119, and in which Cat SPF 9 received negative

384

LIFE CYCLE OF THE COCCIDIAN PARASITE, T O X O P L A S M A

G O N D I I I N THE DOMESTIC CAT

brains, is g i v e n in T a M e I. T h e s e negative brains were taken f r o m m i c e w h i c h had dyetest titres o f < 1 : 5 w h e n e x a m i n e d on b o t h occasions at 30 day intervals. I n contrast, the brains containing the tissue cysts o f strain 119 c a m e f r o m m i c e whose dye-test titres v a r i e d f r o m 1 : 250 to 1 : 1,250 on the 30th day after infection. TABLE I. T h e results obtained by feeding 15 Toxoplasma dye-test negative, S P F cats with either the tissue cysts or o6cysts of Toxoplasma gondii. Cat S P F 5 received no infectious material, while Cat S P F 9 was fed with brains taken f r o m mice which were b o t h serologically and parasitologicaUy negative for Toxoplasma.All tissue cysts used were of the Beverley strain except those fed to Cat S P F 8; in this latter instance, Statens Seruminstitut strain 119 was used. S P F Cats

Toxoplasma Cat Age No. ( w e e k s ) 1

Cat autopsy

Day

Weight (Kg.)

material fed

--

tissue cysts

Toxoplasma o6cysts first shed (N.S. = not shed)

Day

Coccidia forms in gut

Dye-test titre

N.S.

3

<1 : 5

none seen

2

22

6

6

<1:5

all stages

3

14

5

5

<1:5

1 schizont only

4 I

14

o6cysts

N.S.

5

<1:5

none

5

14

none (control)

i'q.S.

6

<1 : 5

none seen

tissue cysts

i0

19

1 : 250

not examined

10

19

1 : 250

not examined

6

14

1 : 250

all stages

7

seen

8

22

4-65

9

22

3'25

neg. brains (control)

N.S.

14

<1 : 5

none seen

10

22

3-10

tissue cysts

N.S.

4

<1 : 5

none seen

11

16

2 "50

7

13

12

16

2'31

5

5

<1:5

all stages

13

16

2 "20

N.S.

6

<1 : 5

none seen

14

20

N.S.

34

1 : 50

not examined

15

25

N.S.

34

1 : 250

not examined

16

25

3"12

tissue cysts

5

6

1 : 10

all stages

17

34

3"76

o6cysts

9

11

o6"cysts

all stages

1 : 250

1:50

I

all stages

W. M. HUTCHISON,J. F. DUNACHIE,K. WORKAND J. CHR. SIIM

385

At Strathclyde, examination of the brains on arrival revealed no tissue cysts in the negative mice but large numbers in the brains of those which were sero-positive. Further, the brain emulsion prepared from the negative mice did not give rise to Toxoplasma infections in the 12 mice into which it was injected. The 12 mice injected with the cystcontaining emulsion, on the other hand, were positive for Toxoplasma,both serologically and parasitologicaUy, when examined 35 days later. These preliminaries may seem overelaborate but we felt that they were essential in order tO establish, firstly, that no mixing of positive and negative brains had occurred and, secondly, that the tissue cysts in the brains sent from Copenhagen were still viable at the commencement of the cat experiment 48 hours after the deaths of their hosts. Cat SPF 8 started to shed o6cysts in the faeces on the 6th day after the infective meal and was killed on the 14th day; its dye-test titre at autopsy was 1 : 250. Histological examination of the small intestine revealed that schizogony and gametogony were taking place in the epithelial cells of the tips of the villi along the entire length of the small intestine. SPF 9, which had been fed with the Toxoplasma-negativemouse brains, was killed at the same time. It was sero-negative, having a dye-test titre of ~ 1 : 5; o6cysts were not seen in its faeces, neither was schizogony nor gametogony observed in the intestinal epithelial cells. Separations from its faeces did not result in toxoplasmosis in the mice to which they were fed.

Cats fed with o~eysts Cats SPF 4, 13, 14, 15 and 17 were each fed with approximately t million o6cysts which had not been sporulated longer than 6 months. SPF 4 and SPF 13 were killed on the 5th and 6th days respectively after feeding in an attempt to detect early stages of the infection. Nothing was observed in the epithelial cells on histological examination; both cats remained dye-test negative and did not shed o6cysts. SPF 14 and SPF 15 were maintained for 34 days after being fed with the o6cysts but during this lengthy period no of cysts appeared in the faeces. Despite this, there is no doubt that both cats were infected successfully because SPF 14 and 15 developed dye-test titres of 1 : 50 and 1 : 250 respectively. Cat SPF 17 shed o6cysts in the faeces 9 days after being fed; it was killed on the 1 lth day. Impression smears revealed that all stages of the parasite were present in the intestine. Histological examination revealed schizogony and gametogony within the epithelial cells, indicating that these processes can be initiated by sporozoite infections as well as by the zoites from the tissue cysts. In conclusion, in all the experiments described above, there were no instances in which o6cyst-negative cat faeces caused Toxoplasmainfections when fed to mice. Toxoplasma infection only occurred, and always occurred, when the mice were fed sporulated o6cysts.

Developmental stages in the epithelium of the gut of the cat The infected tissues which we have examined histologically have shown all stages of the coccidian life cycle. The ileum was found to be the commonest site of infection, although in the case of SPF 8 the entire small intestine was involved. The infection appeared to be concentrated in the cells of the tips of the villi. Within the infected cells, the parasites were found between the nucleus and the brush border. Different stages of the life cycle occurred in no particular sequence along the epithelium and multiple infections of the ceils were common, as is apparent from our illustrations.

386

L I F E CYCLE OF THE C O C C I D I A N PARASITE~ T O X O P L A S M A

G O N D I I I N THE DOMESTIC CAT

r

1

2

5

6

I

0

8 !

liO

20 p

PIGS. 1-8. x 2,000. Development of schizont of Toxoplasma gondii. Carnoy-fixed material, stained with Heidenhain's iron haematoxylin. Detail of host cell nucleus and cytoplasm omitted. FIG. 1. Invasive merozoite within host cell. T h e initial orientation of the merozoite is parallel to the long axis of the host cell; this mfly alter later. PIG. 2. A n invasive merozoite i n process of assuming its ultimate position within the host cell, at right angles 'to its long axis. N o surrounding vacuole is as yet apparent. FIG. 3. Two parasitized epithelial cells. T h e one on the left has a double infection. I m m e d i ately below the brush border of the host cell a trophozoite lies within a vacuole. Below it, also within a vacuole, is a developing macrogametocyte. I n the host cell on the right, a trophozoite has its karyosome i n process of division. PIG. 4. Host cell with double infection. Below the brush border, within a vacuole, is an early, binucleate schizont. T h e karyosome of the nucleus on the right appears about to divide. Below the schizont lies a trophozoite. PIG. 5. 6-nucleate schizont within a vacuole. T h e nuclei lie at different levels within the schizont; each has a prominent karyosome. FIG. 6. 12-nucleate early schizont. T h e nuclei are smaller than those of the 6-nucleate stage, and the karyosomes less prominent; one nucleus is apparently in process of division. Nuclei lie at different levels within the schizont. F I 6 . 7 . Host cell with double infection within a single vacuole. Below the brush border is a trophozoite, perhaps large enough to be considered an early macrogametocyte. Below this, a late schizont with 10 nuclei which are densely staining and peripherally oriented; not indicated by this staining method is the fact that they are now Feulgen-positive. Cf. Plate 1, Fig. 3. Plo. 8. Host cell with triple infection. Directly above the nucleus lies a mature schizont cut in radial section to show the merozoites spread fan-wise. 15 merozoites are visible; there are others which have not come into the plane of section.T h e merozoite nuclei are densely staining with a karyosome visible in most cases. Outside the merozoites there is very little residual material. Above the schizont lies a microgametocyte (cf. Plate 2, Pig. 1, Mil). T o the right of this is a parasite with 2 nuclei visible.

Schizogony The first stage in development which we have observed has been the merozoite which has just penetrated into a cell of the epithelium (text Fig. 1 ; Plate 1, Fig. 4). It is

W. M. HUTCHISON, J. F. DUNACHIE~ K. WORK AND J. CHR. SIIM

387

relatively unchanged in appearance from those found in mature schizonts (text Fig. 8; Plate 1, Fig. 4), but the nucleus is less densely staining and its karyosome is quite distinct. Immediately after penetration, it lies parallel with the long axis of the cell, but ultimately may assume a position at right angles to it, just below the brush border. Here initial development as a trophozoite takes place within a vacuole (text Fig. 3). The organism increases in size, growing in width faster than in length, while retaining its ellipsoid shape; in transverse sections it is circular (Plate 1, Fig. 2). The nucleus alters greatly in appearance, becoming spherical and vesicular with a very prominent karyosome*; until the final stages of schizogony the whole nucleus is Feulgen-negative. Occasionally a faint tinge of colour can be seen in the karyosome and nuclear membrane. The karyosome stains pink with methyl green-pyronin, suggesting the presence there of RNA (PEARSE, 1960), and very densely with Heidenhain's iron haematoxylin (text Figs. 3-6), which also stains the fine nuclear membrane. The granules in the cytoplasm of the merozoite are few and small; they increase in number and in size as the organism grows into a trophozoite. They colour densely blue with Giemsa (Plate 1, Figs. 1-5) and brightly pink with methyl green-pyronin; only a small proportion of them appear to stain with Heidenhain's or Ehrlich's haematoxylin; the cytoplasm is strongly basophilic with both these stains (text Figs. 3 and 4; Plate 2, Figs. 1, 3 and 4). As the organism increases in size, nuclear division takes place, unaccompanied, as far as can be seen under the light microscope, by division of the cytoplasm. The process is evidently a rapid one and must include one or more stages which we have not yet observed. The nucleus elongates and the karyosome divides (text Fig. 3). The two products move apart, presumably to become the karyosomes of the two nuclei which result from the completed division (text Fig. 4). Division of the nuclei continues, and as their numbers increase within the organism they become somewhat smaller and their karyosomes less prominent (text Figs. 5 and 6). They also become more difficult to distinguish and count in sections, lying as they do at different levels within the basophil cytoplasm of the developing schizont. If the Giemsa/ Colophonium method for sections is used, the nuclei are further obscured by the cytoplasmic granules staining densely blue (Plate 1, Figs. 2 and 5). In Giemsa-stained smears, however, nuclear detail is more easily visible (Plate 3, Fig. 2), Ehrlich's haematoxylin, on the other hand, does not differentiate distinctly enough between the karyosome, nuclear sap, and cytoplasm (Plate 2, Figs. 1-4). When they cease to divide, the nuclei become smaller, more densely staining, and Feulgen-positive. The cytoplasm is granule-packed and very basophilic. The nuclei now move to the periphery of the schizont (text Fig. 7), and a portion of cytoplasm becomes associated with each of them (Plate 1, Fig. 3). These associations become more clearly defined and finally separate as merozoites, leaving a residual body (text Fig. 8; Plate 1, Fig. 4). In this terminal stage, the granules and basophilia of the schizont have become much reduced. Its configuration tends typically to be asymmetrical, with the merozoites radiating in three dimensions from a point on the border of the schizont, rather than from its centre. Sections passing through the radial axis of the schizont thus show the merozoites spread fanwise, rather than in a complete rosette (text Fig. 8; Plate 1, Figs. 1 and 4). Merozoites in sections are 3.5-4.5~ long and about l~z in diameter at their broadest point. In smears, due to their flattening by this process, they appear somewhat longer and *A karyosome is usually taken to mean a nuclear inclusion in which DNA can be demonstrated. Consequently, the term could be regarded as incorrect when used here with Toxoplasma. However, the structure appears so obviously homologous with the karyosome found in Isospora spp. that the term has been retained in Toxoplasma.

388

LIFE CYCLE OF THE COCCIDIAN

PARASITE~ TOXOPLASMA

GONDII IN THE DOMESTIC CAT

broader, 4-9~ (SD * ~-- 1"0) by about 1.5~z. This is the only size of merozoite which we have observed at the stage of the infectionwith which we have been working; our measurements from smears are of 200 merozoites and give a fairly normal distribution with no sign of heterogeneity. As has been mentioned, the nucleus of the merozoite stains densely, both with Giemsa and with haematoxylin stains. A karyosome can sometimes be detected and is probably always present. Within the cytoplasm, some of the granules are PASpositive; the developing schizont contains little or no PAS-positive material in contrast to its presence in the developing micro- and macrogametocyte. The number of merozoites resulting from a schizont varies considerably. Table I I is a histogram showing this variation in numbers of merozoites found in 208 schizonts, observed in impression smears. 72% of the schizonts contain between 8 and 17 merozoites, and 61-5% between 10 and 15 merozoites. TABLE II. Variation in number of merozoites per schizont, taken from counts of 208 schizonts in impression smears.

R

Z

4"~-

6-~7

8-9

10*11

12~1~ 14-15_ 16e17 18-19 20~21

22-23J 24-~25" ~6"~Z7. 28°29,

Numberof merozoites per.schizont

Gametogony

The microgametocyte The question of whether the earliest developmental stages of the microgametocyte can be distinguished from those of the schizont is raised in the discussion. The later stages of microgametogony in sections are sometimes difficult to correlate with the same stages in smears because smearing can distort the cells of both host and parasite. The first stage which can definitely be characterized as a microgametocyte is spherical with a diameter of about 10~z (Plate 2, Fig. 2). Its cytoplasm, which is slightly basophilic and contains PAS-positive material, appears to be fissured into a series of bands and *SD = Standard deviation.

KEY Des gob hn Ma’ Ma2 Me. i. Mg

= = = = = = =

TO LETTERING

OF PLATES Mi’ MiZ to Mi” Mi” SC’ SC2 SC3 Tr

degenerating parasite goblet cell host cell nucleus early mac*ogametocyte late macrogametocyte invasive merozoite microgamete

EXPLANATION

Sections

through

the tips of villi in the ileum of a cat infected

= = = = = = =

earliest microgametocyte intermediate microgametocyte

stages

mature microgametocyte early schizont late schizont mature schizont with separated trophozoite

merozoites

OF PLATES

PLATE 1. with Toxoplasmagondii.

Carnoy-fixed

material.

Giemsa/Calophonium

stain.

FIG. 1, Low power view ( x 530); the tip of one villus and parts of others, are shown in longitudinal section. The parasites are readily recognized by the greater affinity of their cytoplasm for the methylene blue component of the stain, compared with that of the host cells. Note the frequent presence of more than one parasite within a host cell in this heavy infection and;;pu,‘x lack of any sequence of developmental stages along the length of the FIG. 2. x 2,125. Developmental granules,

stages lying between nucleus and brush border of host cells, viz., early macrogametocyte with densely early schizont with 6 karyosomic nuclei; trophozoites are cut transversely and longitudinally.

FIG. 3. x 2,125, A triple infection

with 1 early and 2 late schizonts within a single vacuole. In the latter schizonts, merozoite and small, dense, non-karyosomic nuclei have become arranged at the periphery.

FIG. 4. x 2,125. A mature schizont (cf. text, Fig. 9). An invasive

formation

blue-staining is commencing

has been cut along its radial axis so that its merozoites appear to be spread fan-wise around a small residual body merozoite (cf. text, Fig. l), early schizonts, and an early macrogametocyte are also infecting adjacent cells.

FIG. 5. x 2,125. The extreme tip of the villus is at the upper end of this illustration. A mature microgametocyte contains crescentic microgametes and a residual body which is rather larger than usual (cf. Plate 3, Figs. 7 and 8). Also present are 2 late macrogametocytes; the granulation typical of the earlier stage (e.g., Fig. 2) is absent; the vacuoles in the cytoplasm are in fact filled with PAS-positive granules (cf. Platz 2, Fig. 3). Note the apparent activity of the karyosome of the larger macrogametocyte. Amongst other stages present are an early schizont with nuclet obscured by granules 2nd a mature schizcnt with merozoites cut transversely. There are degenerating forms at the extreme tip of the villus where epithelium is shed.

PLATE 2. x 2,000. Material as in Plate 1, but stained with PAS for polpsaccharides and Ehrlich’s haematoxylin. Macro- and microgametocytes are the most prominent and the presence of PAS-positive material in them contrasts with its apparent absence in the schizonts illustrated. A single host cell nucleus (hn) is indicated in Fig. 1; the others czn be distinguished by comparison with this. FIG. 1. The developing macrogametocyte has PAS-positive granules not yet filling the cytoplasm; extrusions from its karyosome suggest that this is metabolically active. The nucleus and cytoplasm of the trophozoite and schlzont, present in adjacent cells, cannot be well differentiated. FIG. 2. Earliest identifiable sage of the developing microgametocyte (cf. Plate 3, Fig. 4). One individual is circular in section and represents the typical spherical form) the shape of the other has been distorted. Nuclei are not clearly distinguishable. A fragment of a late schizont is apparent in the section with ridges where the merozoites are in process of formation. PAS-positive mucus is present in a goblet cell. FIG. 3. T.S. of villus, near its tip; host ceil nuclei have been displaced and somewhat distorted by the parasites. Several stages in the development of the microgametocyte are to be seen, viz., the earliest identifiable stage (Mi’), the stage showing the nuclei becoming peripherally oriented (Mil), and that which shows the begmning of the elongation of the microgametes (MP). A mature microgametocyte is also present (MP). FIG. 4. Microgametocyte with nuclei peripherally arranged and surrounded with patches some of the nuclei indicate that development of the elongate microgamete has commenced.

of clear cytoplasm An early schizont

(cf. Plate 3, Fig. 6). Protrusions from lies above an obliquely-cut goblet cell.

PLATE 3. x 2,050. Ftcs.

1-7, impression smears from ileum of an infected cat, Bouin-fixed and stained with Giemsa, to show developmental (Figs. l-3) and microgametogony (Figs. 4-7). Fig. 8, a section from Carnoy-fixed material, stained Feulgen’Fast

FIG. 1. Trophozoite

and a binucleate

stage where,

FIG. 2. Triple

infection

with

FIG. 3. Mature

schizont.

17 merozoites

FIG. 4. Early

microgametocyte

within

the vesicular nuclei, granules.

the karyosomes

appear

to have been ruptured

2 trophozoites and an early 8-nucleate schizont (cf. Plate 1, Fig. 2). The vesicular apparent; the shape of the karyosomes seems to have been distorted by smearing.

(=

Mi’)

have been formed with

6 nuclei

nature

and dispersed

of the schizont

lying within strands of cytoplasm invasive merozoite.

(cf. Plate 2, Fig. 2 and 3). Also within

( = Mi2) with nuclei apparently comp1etin.g division. The orientation of nuclei within by smearing. Also vi&in the host cell is a trophozoite.

FIG. 6. Microgametocyte

(= MP)

the organism

(=

Mi”)

nuclei is

with

17 microgamates

(Mg)

and a small residual

FIG. 8. Section through a mature microgametocyte (= MP), stained Feulgen/Fast green, to demonstrate complete length of the body of the microgamete. A trophozoite with a Feulgen-negative, karyosomic nucleus section.

body.

by

the host cell is an

may have been distorted

with 31 developing microgametes; their peripheral orientation is not apparent due to the nature smearing. Cf. Plate 2, Fig. 4. Note the apparent absence of any residual body.

microgametocyte

into

but their configuration (cf. Plate 1, Fig. 4) has been lost and they have been flattened the process of smearing.

FIG. 5. Microgametocyte

FIG. 7. Mature

stages of schizogony green.

of the process

of

Cf. Plate 1, Fig. 5.

that the nucleus extends virtually the can be seen (above, R) cut in transverse

PLATE 1 SC” Ma’

SC’

Mi’

Ma’

1

Tr

SC?

0.

SC2 IO3 hn

Me.i.

Sc3

Tr

SC’ Ma’

SC’

n-

PLATE 2 Tr

SC’

Ma’

gob

SC’ hn

1

2 8

I

0

Mi”

Mi’

SC’

Tr

Ma2

Mi4

SC’

Ma2

IO

Ma2

Tr

20 P

Ma’ Tr

Mi3

Tr

Nii’ 3

4

SC’ Tr

Ma’

IFD”NAMlE

PLATE

3

6 hn 10

W. M. HUTCHISON, J. F. DUNACHIE, K. WORK AND J. CHR. SlIM

389

wedges. Within it, nuclear material, which is Feulgen-positive, appears to be distributed in rather diffuse patches. The appearance of this stage in Giemsa-stalned smears is shown in Plate 3, Fig. 4. The nuclear material next becomes more compact and tends to assume a peripheral position; the cytoplasm appears less fragmented while its content of PAS-positive material increases (Plate 2, Fig. 3). In smears (Plate 3, Fig, 5), the peripheral alignment of the nuclear material is lost; its appearance suggests that it is undergoing, or has undergone, division. The next stage (Plate 2, Fig. 4) shows completely compact nuclei lying in clear patches of cytoplasm, mainly at the periphery of the organism. From these nuclei, small rod-like structures may be seen to project; ultimately these elongate to form the slender microgametes. This stage can also be seen in smears (Plate 3, Fig. 6) where, again, the peripheral alignment of the nuclei is lost. The mature microgametocyte shows considerable variation both in the number of its microgametes (between about 12 and 32) and in the quantity of its residual material. The microgamete is (excluding its flagella) about 3~ in length and tends to be crescentic in shape (Plate 1, Fig. 5; Plate 3, Fig. 7). The nucleus extends for almost the whole length of the microgamete, as is shown by the Feulgen reaction (Plate 3, Fig. 8). The microgamete appears, under the light microscope, to be pointed at both ends.- The flagella are not readily seen under the light microscope but are quite distinct in our electron micrographs. PIEKARSKI(1971) has demonstrated a third, rudimentary flagellum. The macrogametocyte The development of the macrogametocyte appears to be relatively simple, with its growth in size as its most obvious feature. An organism that has attained dimensions greater than about 7 by 5~ without nuclear multiplication can be assumed to be a developing macrogametocyte. The nucleus, while increasing in size, retains, fundamentally, its initial structure and staining properties during the whole of development. The karyosome, the only structure appearing within it, is a spherical body which comes to be surrounded, as development proceeds, by less dense material which, in sections, appears to cap it (text Fig. 3; Plate 1, Fig. 4). It stains strongly with Heidenhain's, but weakly with Ehrlich's haematoxylin. The presence of RNA within it is indicated by its staining pink with methyl green-pyronin. The nucleus remains Feulgen-negative throughout the course of development, with the exception of an occasional line of colour at the nuclear membrane. Changes occur in the staining properties of the numerous granules within the macrogametocyte during the course of its development. Until a fairly late stage, granules remain which stain densely blue with Giemsa (Plate 1, Figs. 2 and 4). From almost the earliest stages granules which are PAS-positive can be seen, though it cannot be certain whether or not they are identical with those already mentioned. As the macrogametocyte matures, granules taking up blue with Giemsa decrease until none remain (Plate 1, Fig. 5). The PAS-positive granules increase in number and size until the cytoplasm is packed full of them (Plate 2, Fig. 3). Among all the granules seen, none appears to be especially associated with the formation of the o/Scyst wall, as do the "plastic granules" of some other species of Coccidia, e.g., Eimeria tenella and Isospora rivolta. Fertilization has not been observed. Since we have seen no micropyle through which a microgamete could pass, we are assuming that fertilization takes place prior to the formation of the cyst wall. The o6cyst, which the zygote has now become, remains for a time in the epithelium before being shed into the lumen of the gut. In freshly deposited stools, o/Scysts have never been seen to be sporulated; on the basis of the measurements of 50 individuals, they have dimensions of 12.7 (SD = 0.7),~ by 10.4 (SD = 0.8)~. They are generally completely filled with protoplasm; after a period of incubation in the external

390

LIFE CYCLE OF THE COCCIDIAN PARASITE, T O X O P L A S M A G O N D I I I N THE DOMESTIC CAT

environment, this retracts to form the primary sporoblast. In sections and smears from the cat intestine, a similar retraction from the cyst waU can be seen. This, however, is probably due to fixation shrinkage. On the completion of sporulation, 50 sporocysts gave measurements of 7.8 (SD = 0.9)~ by 6 (SD = 0.5)~t.

Discussion The establishment of the Medical Research Council's SPF cat colony has been described in detail by BLEBY and LACF.Y(1969). This excellent paper gives an indication of the meticulous care which had been taken to ensure that the colony members are free of all known feline pathogens. In fact, this is probably the only breeding colony of SPF cats in the world which is free of the highly infectious feline respiratory viruses. However, the presence or absence of viral infections in our cats is irrelevant to the project under discussion. Obviously, it is an asset for any experimental animal to be free of such organisms but apart from this, if the cats are being kept under conditions which prevent them from contracting these highly infectious agents, then it is highly probable that other agents, such as Coccidia and toxoplasms;will also be excluded. The cats in the SPF colony are hysterectomy-derived animals and are therefore free of all organisms which are incapable of trans-placental passage. This would include all Coccidia with the exception of Toxoplasma which can cross the placental barrier. However, the negative results obtained when the breeding queens were dye-tested excluded the possibility of the congenital transmission of toxoplasmosis within the colony. The cats are kept under high standards of management and in complete barrier buildings. The nature Of the barrier preventing the entry of pathogens into the colony is described by HILL (1966). All animals are kept under close surveillance and, during the 4 years of the colony's life no cat has shown clinical signs of any recognized feline infectious disease. No evidence of any enteric parasites has been obtained in the weekly faecal samples which are cultured and also subjected to flotation techniques. An adult cat is sent to the laboratory every 6 to 8 weeks for extensive histopathological and microbiological screening. The complete alimentary tract is subjected to a detailed parasitological examination. None of these tests have yet revealed a positive result (BLACKMORE,1970, personal communication). On this basis, we can repudiate any charges that the use of adult SPF cats has provided us with results which are inconclusive. We have shown that the SPF cats which we have used were free of Coccidia and 'Toxoplasma. Consequently, the only explanation of o6cysts in the faeces is that they, and the intermediate forms present in the intestinal epithelium, are related to the Toxoplasma tissue cysts which were fed to the cats. C a t s fed with tissue cysts (a) Beverley strain It is possible that Cat SPF 1 was examined before the Toxoplasma stages became patent in the epithelial ceils of the small intestine. However, if the organisms are not observed before the 6th day in either the faeces or on histological examination, as was the case in SPF 1 and SPF 10, it is impossible, because of the absence of serological evidence, to decide whether the toxoplasms which have been administered have failed to establish themselves in the host or whether they are still in a prepatent phase. Even where Toxoplasma can be detected by faecal and histological examination it is our experience that the cat remains sero-negative for about 6 days after the toxoplasmic meal (Table I). Cat SPF 2 was ~ 1 : 5 at 6 days after feeding tissue cysts although the titre of Cat SPF 16

W. M. HUTCHISON~ J. F. DUNACHIE, K. WORK AND ~. CHR. SIIM

391

had risen from < 1 : 5 to 1 : 10 over the same period. This may indicate that seroconversion occurs around the 6th day after ingestion of the infective meal but obviously more data would be required to confirm this. (b) Statens Seruminstitut strain 119 Cat SPF 8 started to shed o6cysts on the 6th day after feeding with strain 119. By permitting the experiment to run for 14 days, we obtained serological evidence of a Toxoplasma infection, as the dye-test titre had risen from < 1 : 5 to 1 : 250. No seroconversion was observed in the control cat (SPF 9) fed with negative brains. This experiment with SPF 8 and 9 established that the coccidian o6cysts observed in the faeces of cats fed with mouse brains were related to the Toxoplasmainfections which were contained in the brains and not to other hypothetical brain infections, such as unknown coccidian stages. In addition, it confirmed the earlier control experiment with SPF 2, 3, and 5 by producing additional evidence based on serology that the infections which we were observing in the faeces and in the intestinal epithelium were related to Toxoplasma. FRENKEL et al. (1969) considered that strain M-7741 which they used was more efficient in producing the faecal forms (i.e., o6cysts) of Toxoplasmathan were others, including the Beverley strain. We ourselves were certainly very impressed by the results obtained using the strain 119 in SPF 8; the entire length of the small intestine of this cat was infected. In our present experience, this seemed to compare favourably with the Beverley strain in which the infection appears to be restricted to the ileum. However, much more work would be required to establish that particular strains are superior to others in their ability to infect cat gut epithelium. Cats fed w i t h o~icysts There are two possibilities for the negative results which we obtained with SPF 4 and 13. The first is that we failed to infect the cats with the o6cysts which were used. This cannot be confirmed serologically as, in our experience with tissue cyst infections, dyetest antibodies are not always apparent even in an established infection of 5 to 6 days duration. Another possibility is that we were successful in infecting the cats but that they were killed before the infection could be demonstrated either serologically or histologically. In SPF 14 and 15 we again failed to produce o6cysts. Due to the results reported by FRENKELet al. (1970), we allowed the experiment to run for 34 days. At autopsy, the dye-test titres gave clear indications that sero-conversion had taken place. We take this to mean that cats, in common with many other animals, sometimes can become infected with Toxoplasma o6cysts without the parasite producing the gametogonic stages. Since Toxoplasma antibodies were absent from these cats on infection, it is evident that this factor could not interfere with o6cyst production in this instance. The work of FRENKELet al. (1970) has shown that when cats were fed with Toxoplasma o6cysts, it required 21-24 days before a new generation was produced. Our single positive result (SPF 17) differs in that the infection produced o6cysts on the 9th day but to what extent this can be attributed to the different strain which we used is unknown. Others (PIEKARSKI,1970) have also induced o6cyst production at such an early stage by feeding o6cysts.

Toxoplasrna to Isospora spp. o f cats o6cyst of Toxoplasmawas shown to be disporocystic and tetrazoic by

Relations of

The SIIM et al. (1969). The observation by HUTfiHISONet al. (1969a, 1970) and FRENKELet al. (1970) of schizogonic cycles and gametogonic stages in the intestine of the cat gave clear indication of the coccidian nature of Toxoplasma. It thus became possible to classify Toxoplasma

392

LIFE CYCLE OF THE COCCIDIAN PARASITE, T O X O P L A S M A

G O N D I I I N THE DOMESTIC CAT

under the Sub-phylum Sporozoa, Class Telosporea, Sub-class Coccidia, Order Eucoccida, Sub-order Eimeriina. The resemblance of the o6cyst of Toxoplasma to those of the genus lsospora is already well known. In particular, the existing descriptions of the o6cyst of Isospora bigemina (small form) seem applicable to the o6cyst of Toxoplasma. It may well become apparent in the future that Isospora bigemina and Toxoplasma gondii are names which refer to the same organism. Should this prove to be so, the trivial name, bigemina Stiles 1891, Liihe 1906, should have priority over that ofgondii Nicolle and Manceaux 1908. At this point we feel we ought to make it clear that, at present, we are entirely opposed to any alteration to the nomenclature of Toxoplasma gondii. Any alteration of the generic name (e.g., to lsospora) should lead to a similar alteration in the name given to the disease which is caused by the organism.This we feel would be an unnecessary play upon words and would almost certainly lead to confusion amongst clinicians and others who are not concerned with these finer points. Article 23, (a) (ii) of the International Code of Zoological Nomenclature (1961) makes provision for the retention of well known names which have been in general use for a considerable period of time, irrespective of priority. Apart from this, GARNHAM(1970) has pointed out that a coccidian with an o6cyst which contains 2 sporocysts, each with 4 sporozoites, does not necessarily qualify automatically for inclusion in the genus lsospora. HOARE(1956) and LEVINF-(1961) in their tables of coccidian classification based on o6cyst morphology make it clear that more than one genus can occupy a single morphological pigeon-hole. For example, the genera Pfeifferinella, Schellackia and Tyzzeria are all placed together in the asporous, octozoic group of the Eimeriidae. The decision with regard to taxonomic changes, however, must be influenced by the ultimate elucidation of the life cycles of Besnoitia, Sarcocystis, and Frenkelia, which are considered to be closely related to Toxoplasma. In addition, this decision may be influenced by the fact that no species of Isospora, as far as has been reported, exhibit either endodyogeny or tissue cyst formation. Indeed, if odd instances occur where this is observed, there may well be a case for including the relevant species under the genus Toxoplasma rather than vice versa. Decisions will also have to be taken with regard to the naming of specific stages in the life cycle but this aspect will be discussed subsequently. The dimensions of the o6cyst of Toxoplasma, as ascertained by ourselves and other workers, correspond to the fairly wide limits of the measurements given for Isospora bigemina by WENYON (1926) (see Table III). There appear to be a "large" and a "small" form or variety of lsospora bigemina, each with a considerable range of size, though with no overlap between them. The o6cyst of Toxoplasma corresponds to the measurements given for the "small" form of I. bigemina. Should they in fact prove to be identical, i t m a y be possible to reserve Isospora bigemina as the pro parte name for the "large" form and retain the name Toxoplasma gondii for the "small" form. This procedure would still lie within the International Code of Zoological Nomenclature, but before it could be adopted, the situation with regard to the lsospora of the cat, and in particular that of lsospora bigemina, should be clarified. WENYON (1923, 1926) is the only author to give descriptions of the earlier stages of I. bigemina in the gut of the cat; to equate them with the stages of Toxoplasma presents difficulties. In the first place, Wenyon seems to have almost invariably found them, not in the epithelium, but within the lamina propria of the intestinal villi, where all stages, including sporulated o6cysts, could be seen. He conceded that these parasites might in fact be stages of lsospora rivolta developing atypically in the lamina propria, though MAHRT (1967) describes this as the typical site for this species. This could account for the fact that the o6cysts were of the "large" variety of I. bigemina whose sporocysts have

W. M. HUTCHISON, J. F. DUNACHIE~ K. WORK AND J. CHR. SIIM

393

dimensions identical with those of I. rivoha. Paradoxically, however, in his illustrations, WENYON (1923, 1926) has shown both oScysts and sporocysts of the "small" variety in the lamina propria. T h e earlier stages, particularly the microgametocyte, are indeed similar to those we have found for Toxoplasma, but are only about half their dimensions. For example, the microgametocyte is shown as having a diameter of about 5~t, as against about 10p for Toxoplasma. TABLE I I I . Comparison of o6cyst and sporocyst dimensions of Isospora bigemina with those of Toxoplasma gondii. Mean values are given, except for Wenyon, where only the range of sizes is available. o6cyst (~)

sporocyst (~)

18-20 × 14--16 10-16 × 7-5-10

13.5-15.5 × 9-10 7.5-10 × 5-8

Isospora bigemina WENYON (1926) "large" "small" LEVINE & IVENS (1965)

11-7 × 10.4

Toxoplasma gondii OURSELVES(p. 390)

12.7 × 10-4

7.8 × 6 ' 0

FRENK~.L et al. (1970)

12

about 8 × 6

OWRHULVE (1970)

11-2 × 10-4

SHEFFIELD & MELTON (1970)

12

WEILAND ~= K U H N (1970)

WITTE & PIEKARSKI(1970)

× 10

×

9

12 ± 1-0 × 10 ± 0-8 7.4 ± 1.0 × 6.1 ± 0.7 12-6 × 10-7

*WORK ~ HUTCHISON

(1969 a, b)

14

×

9

7×3

*This material has been re-examined and on the basis of observations on 50 o6cysts, the mean measurements are now given as o6cyst: 12.8 × 9.91~; sporocyst: 7.4 × 5"51x. In the second place, only one example of I. bigemina infecting the intestinal epithelium has been reported (WENYON and SHEATHER, 1925; WENYON, 1926). This is of an infection found in a dog and unfortunately is not illustrated. Numerous macrogametocytes are mentioned as being present and are stated as having a diameter of 7.5~t, which is about that of the long axis of immature macrogametocytes of Toxoplasma which we have observed. Mature schizonts of I. bigemina are stated to be not more than 5~ in diameter and to form 8 "minute" merozoites, whose size, unfortunately, is not given. Schizonts of 5~ in diameter, while on the small side for Toxoplasma, are certainly large enough to give rise to 8 merozoites. I f we allow for the possibility that there may be differences in detail between the stages in the infection in the dog compared with that in the cat, we cannot deny the possibility that this infection was in fact of Toxoplasma. Isospora bigemina is also reported to infect foxes, polecats, and mink (LEVINE, 1948, 1961) besides cats and dogs. It is to be hoped, and assumed, that natural infections producing oScysts in all these hosts will be investigated. More detailed descriptions are available of Isospora felis, whose infection is confined to the intestinal epithelium of the cat. Accounts of schizogony and gametogony have been

394

LIFE CYCLE OF THE COCCIDIAN PARASITE, T O X O P L A S M A

G O N D I I I N THE DOMESTIC CAT

given by WENYON (1923, 1926), HITCHCOCK (1955), and LICKFELD(1959). There are sufficient resemblances between the developmental stages of I. fells and Toxoplasma to make a comparison of these two different species useful. The resemblances may perhaps assist us in clarifying structures within the different stages of Toxoplasma. The differences seem to be due largely to the fact that in all its developmental forms I. fells is a far larger species. According to Lickfeld, mature schizonts measure approximately 38 × 20~z, giving rise to merozoites of 2 sizes at different stages, viz., 16 -- 18-5 × 5 -- 8~z and 7.5 x 2-5~. Microgametocytes are approximately 72 x 60~ and oOcysts measure 43 × 32~. Thus it causes obvious deformation and damage in the cells it infects; the cytoplasm of the host cells is hypertrophied and the nuclei almost obliterated. Toxoplasma, on the other hand, even in a multiple infection, appears to be accommodated relatively comfortably. The host cells retain their normal dimensions and their nuclei though not completely unaffected by the presence of the parasites, appear to be relatively unaltered during the stages of the infection which we have examined. Viewed under low power microscopic magnification, the epithelium is perfectly regular, with its parasites, even in heavy infections, barely detectable by conventional staining methods. From the description by the three authors mentioned, the early stages of schizogony oflsosporafelis, though larger, are very much like those we have seen in Toxoplasma; both are typically coccidian. The nucleus is spherical and vesicular, with a large karyosome, while the cytoplasm is granular and densely basophilic, obscuring the nuclei in the later stages. These are all characteristics we have described as typical of Toxoplasma. WENYON (1923) described nuclear division commencing with that of the karyosome, the division of which we have also seen occurring in Toxoplasma (text Fig. 3). He reported the presence of chromosomes arising between the newly-formed karyosomes2 LICKFELI)(1959), however, was unable to find them, and we have seen none in Toxoplasma. On the evidence we have, therefore, nuclear division in the schizont of Toxoplasma is similar to that of I. fells, and is what B~LA~ (1926) characterized as cryptomitotic--i.e., without disintegration of the nuclear membrane or the formation of poles and spindle fibres2 The elucidation of the processes of nuclear division, however, will probably more easily be brought about by electron, rather than by light, microscopy. In contrast to our findings with Toxoplasma, LICKFELD(1959) reports the karyosome of I. fells to be Feulgen-positive in all forms except the later stages of the development of the macrogamete. There seems to be no very good explanation for this difference, unless it indicates a greater activity of the DNA in Toxoplasma. Such activity, by dispersing the DNA and surrounding it with metabolites, would mask it from the Feulgen reaction. Differences emerge when the later stages of schizogony in the two species are compared. The mature schizonts and merozoites are both, of course, much larger in I. felis than in Toxoplasma. LICKFELD (1959) described the formation of "cytomeres" in the schizont, from which the merozoites develop; this can perhaps be regarded as a meroblast stage; it is not present in Toxoplasma. He also demonstrated two types of merozoites, with the different dimensions already quoted. The larger form, or macromerozoites, were formed from schizonts early in the infection; they did not arise from cytomeres and gave rise only to schizonts. The small form, or micromerozoites, were produced later in the infection from schizonts with a cytomere stage, and could develop into gametocytes. We ourselves, with Toxoplasma, have never found merozoites of any but one size, 4-9 B long. In this respect, however, it is of interest that WEILAND and KUHN (1970), also working with Toxoplasma, report, in addition, merozoites with a length of 7.1B 4-0.9. It is possible, therefore, that there are 2 phases of schizont generations in Toxoplasma which give rise to merozoites of different sizes.

W. M. HUTCHISON, ]. F. DUNACHIE, K. WORK AND J. CHR, SIIM

395

The microgametocyte of Toxoplasma is unusual among those of the Coccidia in producing comparatively few microgametes. In sections up to 6~ thick, we have never seen more than 25; even in smears the count lies between 18 and 32. It seems unlikely that the total can ever be more than 40. In contrast, the microgametocyte of Isosporafelis produces about 2,000 microgametes according to WENYON (1923) and between 600 and 1,200 according to LICKFELD (1959). This disparity is less anomalous than it seems, however, when we take into account the fact that the microgametocyte of L fells has a volume approximately 200 times greater than that of Toxoplasma, while producing microgametes about the same size, 3.5~ long. Approximately 50-70 microgametes are found in Isospora rivolta. They are larger (5.8-6.8~ long) but are formed in a microgametocyte whose dimensions (13.4 × 8.7~) are similar to those of Toxoplasma (MAaRT, 1967). The small number of microgametes in Toxoplasma, however, poses the question of whether the early stages ofmicrogametocyte development can be distinguished from those of schizogony. The ultimate number of microgametes per microgametocyte is of the same order as that of merozoites per schizont. Thus, the first stage of what with certainty can be called a microgametocyte in Toxoplasma is spherical, fissured, and has nuclear material distributed through it in a somewhat diffuse state (Plate 2, Fig. 1; Plate 3, Fig. 4). A similar condition in I. felis at this stage is described by LICKFELD(1959), though not by WE~ON (1923). The next stage of Toxoplasma shows nuclear material becoming concentrated on the surface of the microgametocyte (Plate 2, Fig. 3); LICKFELD (1959) reports mitoses at this stage in I. felis. The difference between the microgametocytes of the two species at this stage lies in the fact that in L fells the surface of the organism has been increased by infolding. Such infolding is distinct from the fissures found in the earlier stage of both species. The amount of PAS-positive material increases during development in Toxoplasma; although its occurrence has not been studied in L felis, WAGNER and FOERSTER (1964) report a similar increase in the microgametocyte of Eimeria tenella. In Toxoplasma there appears to be some reduction in the amount of PAS-positive material during and after microgamete formation. LICKFELD(1959) describes considerable activity in the karyosome of the macrogametocyte of I. felis, with particles separating from it and passing through the nuclear membrane into the cytoplasm. The fact that we have not observed a phenomenon of this type in Toxoplasma may simply be due to the fact that it occurs on a much smaller scale. The karyosome, as we have mentioned, and as it appears in our illustrations (text Fig. 3; Plate 1, Fig. 5; Plate 2, Figs. 2 and 3) certainly appears to be active, and its products may well pass into the cytoplasm of the growing macrogametocyte. The significance of the changes inthe staining properties of the cytoplasmic granules is as yet obscure, but the final accumulation of PAS-positive granules probably denotes, at least in part, the storage of material necessary as a reserve for the survival of the o6cyst during the interval spent in the environment before ingestion by another host. That this material is not glycogen is proved by the fact that the reaction of the granules with PAS is not abolished by their pre-treatment with diastase (PEARSE, 1960). In addition to the taxonomic questions which have to be resolved by these new observations on Toxoplasma, there are also problems related to the naming of stages in the life cycle. Most of the new stages, such as schizonts and gametocytes which have been uncovered present no problems as they fall into already well-defined categories firmly established in coccidian terminology. The main problems which arise are whether we can retain the existing names of "trophozoite" (of the pseudocyst) and zoite (of the tissue cyst) in the light of recent developments. Apart from the fact that the term trophozoite must be regarded as pre-empted for the growing, pre-schizogonic, coccidian stage, there

3 9 6 LIFE CYCLE OF THE COCCIDIAN PARASITE, T O X O P L . 4 S M A G O N D I I I N THE DOMESTIC CAT

appear to be no counterparts for them in coccidian terminology. The behaviour of developing pseudocystic "trophozoites" is akin to that of the coccidian merozoites in that both rupture the host cell in which they are developing and escape to parasitize others. The behaviour of the zoites and merozoites is also similar in that both can penetrate the intestinal cells of the cat and initiate schizogony followed by gametogony. However, the zoites differ from the merozoites in that they develop wffhin a cyst membrane which remains intact and which is thought not to rupture with regularity. Moreover, "trophozoites" and the zoites which develop within pseudocysts and tissue cysts respectively, cannot strictly be considered as merozoites as long as they are thought to be products Of endodyogeny and not schizogony. Alternatively, some may be Prepared to refer to either the "trophozoite" or zoite as merozoites by arguing that endodyogeny is a modified type of schizogony. This idea was put forward by JACOBS (1967). Reconsideration may be in order now that schizogony has been described in the life cycle of Toxoplasmaand, as far as light microscope observations are concerned, has been found to be similar to that of other Coccidia. SHEFFIELD(1970), in an electron microscope study~ is in agreemelft that Toxoplasmaundergoes schizogony in the intestinal epithelium of the cat. PIEKARSKI(1971) and his co-workers have made the interesting observation that a process of "endopolygOlly" or multiple internal budding is also taking place. It has generally been accepted that sexual development of Coccidia takes place only in the gut of the host, and certainly the properties of the o6cyst appear to be an adaptation to faecal transmission. However, SHELTONet at. (1968) have described coccidian schizonts, merozoites and gametocytes within macrophages of granulomatous lesions in the dermis of a dog. Of even greater interest is that the dimensions of these stages, as well as the numbers of merozoites per schizont, are consistent with those of Toxoplasmaas we have described them above, though the merozoites are somewhat larger (5.5-6~). It is intriguing, in retrospect, to find GREY (1968) in his comment on this report, suggesting the possibility of a Toxoplasmainfection on the grounds that Coccidia occur only in the small intestine, while SHELTON(1968) replies that Toxoplasmamust be excluded because only asexual reproduction was known to exist in this parasite. The fact that no o6cysts were present in the infection tends to confirm the suspicion that the dermis is not a locale likely to favour the production of resistant and transmissible forms. Yet the findings of SHELTOR!el at. (1968) should make us cautious of declaring the impossibility of a sexual phase of Toxoplasma occurring outside the gut. It is well known that benign strains of Toxoplasmamay become virulent if passaged at short intervals in labaratory rodents, a change which it is presumed is not brought about by a sexual process. Now that a sexual phase has been discovered taking place in the gut of the cat it is possible that we shall encounter more genetic variation than ever before. We have been watching to see whether such a genetic change will be accompanied by an increase in the virulence of the strains we have used. So far no firm evidence of this exists. We suspect that the lethality of our inocula to infected mice (HuTcHISON et at., 1968; WORK and HUTCHISON, 1969a) may have been due to feeding larger numbers of o6cysts than a mouse can tolerate, since similar severe mortality did not persist on serial passages of the infected mouse tissues. Even if the risk of virulent strains arising from sexual reproduction of Toxoplasma in cats or other animals is discounted, there remains a danger that laboratory workers will not realize that they are now dealing with a developmental stage with resistant properties far in excess of those possessed by the stages with which they are more familiar, viz., the "trophozoites" and the zoites. Consequently, much stricter laboratory safety precautions are indicated.

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There is one very interesting question which arises as a result of the recent findings. Has Toxoplasmaany antigenic overlap with the various species oflsospora? In other words, are the serological surveys for Toxoplasma dye-test antibodies accurate or are there crossreactions with the various species of Isospora? No such reaction has been observed with other protozoan parasites, such as Leishmania and Trypanosoma cruzi which also form' colonies in the tissues. To our knowledge, no one in the past ever thought of investigating Coccidia. In this connection there may be a correlation between the high rate of isosporosis (MEEROVlTCHand GIBBS, 1969) and the prevalence of Toxoplasma antibodies (MORALES et al., 1961) in the natives of Easter Island. This is one aspect of human epidemiology which might be worth further investigation. Work now in progress indicates that the sera from cats infected with the o6cysts of Isosporafells are negative for Toxoplasma dye-test antibodies (OwEN, 1970, persofial communication).

Summary Earlier work in which the coccidian nature of the protozoan parasite Toxoplasma gondii was revealed has been confirmed and extended in a series of experiments using only specific pathogen free (SPF) cats. All of these cats, prior to our experiments, were negative for both Coccidia and Toxoplasma. Experiments on 17 of these cats are described. 10 were fed with mouse brains containing tissue cysts of Toxoplasma. 8 of these shed Toxoplasma o~Scysts in their faeces within 5 to 10 days of infection. 2 cats had shed no o/Scysts before they were killed on the 3rd and 4th day after infection. 5 cats were fed with Toxoplasma o6cysts but gametogonic development restflting in the production of o6cysts was observed in only 1 individual; o~Scystsappeared in the faeces 9 days after the infective meal. 2 cats were examined as controls; I received no addition to its diet, the other was fed "negative" brains. Neither showed any evidence of infection. Examination of the gut of infected cats showed that schizogonic and gametogonic stages develop usually in the ileum although the entire length of the small intestine can be affected. The parasites were observed in the epithelium of the tips of the villi, developing within vacuoles, and lying between the nucleus and brush border of the cell. All stages of schizogony and gametogony may be found together, occurring in no particular sequence along the epithelium of the villus; multiple infections of the host cells are common. Schizonts are characterized by granular, basophil cytoplasm and vesicular, karyosomic, Feulgen-negative nuclei which become dense and Feulgen-positive when merozoite formation takes place. The rnerozoites vary from 4 to 29 per schizont and measure 3.5-4.5~ by ll,t in sections; in smears their dimensions are 4.9 (SD : 1.0)~ by 1.5~. They form clusters about a residual body and may appear to radiate fan-wise from it in sections. The microgametocyte is spherical, about 10~ in diameter and gives rise to 12-32 microgametes which form on its surface, are slender and crescentic and about 3~ in length, excluding the flagella. The macrogametocyte has, throughout development, a vesicular, karyosomic and Feulgen-negative nucleus. At the commencement of development, the cytoplasm contains many basophil and few PAS-positive granules; at its completion this condition is reversed. No granules were seen to be associated with the formation of the o6cyst wall. The o6cyst itself measures 12.7 (SD : 0.7)~ by 10.4 (SD : 0.8)~. Sporulation, which does not occur within the gut of the cat, gives sporocysts measuring 7.8 (SD : 0.9)~ by 6 (SD : 0.5)~. These coccidian stages of Toxoplasma are compared and contrasted with those of lsospora spp. of the cat. Despite its significant resemblances to Isospora~the retention of the existing nomenclature of Toxoplasmagondii is supported.

398 LIFECYCLEOF THE COCCIDIANPARASITE,TOXOPLASMA GONDII IN THE DOMESTICCAT REFERENCES AAGAARD,K. (1960). Human Toxoplasmosis (Ed. J. Chr. Siim). Copenhagen: Munksgaard. B~LA~, K. (1926). Der Formwechsel der Protistenkerne Jena: Gustav Fischer Verlag. BLEBY, J. & LACEY, A. (1969). ft. small Anita. Pract., 10, 237. BRAY, R. S. & GARNHAM,P. C. C. (1962). Indian ft. Malar., 16, 153. FRENKEL, J. K,, DUBEY, J. P. & MILLER, N. L. (1969). Science, N . Y . , 164, 432. ,- &(1970). Ibid., 167, 893. GARNHAM,P. C. C. (1970). Proceedings of the Symposium on the present state of Toxoplasmosis Research. Leipzig 1970. GREY, R. M. (1968). ft. Am. vet. reed. Ass., 152, 1084. HILL, A. (1966). ft. Inst. anita. Techns., 17, 133. HITCHCOCK, D. J. (1955). ft. Parasit., 41, 383. HOARE, C. A. (1956). Revta bras. Malar., 8, 197. HUTCHISON, W. M. (1967). Trans. R. Soc. trop. Med. Hyg., 61, 80. , DUNACHIE,J. F., SLIM, J. CHR. & WORK, K. (1969a). Br. reed. ,7., 4, 806. , & WORK, K. (1969b). Syrup. Br. Soc. Parasit., 7, 51. --, - - , SIIM, J. CHR. & WORK, K. (1970). Br. reed. ft., 1, 142. , - & WORK, K. (!968). Acta path. rnicrobiol, scan&, 74, 462. JACOBS, L. (1967). Adv. Parasit., 5, 1. LEVlNE, N. D. (1948)..7. Parasit., 34, 486. (1961). The Protozoan Parasites of Domestic Animals and of Man. Minneapolis, Minn. : Burgess Publishing Company. ---& IVENS, V. (1965). ft. Parasit., 51, 859. LICKFELD, K. G. (1959). Arch. Protist., 103, 427. MAHRT, J . L . (1967). ft. Protozool., 14, 754. MEEROVITCrI, E. & GIBBS, H. C. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 370. MORALES, A., MOSCA, A., SILVA, S., SIMS, A., THIERMANN, E., KNIERIM, F. • ATIAS, A. (1961). Bol. Chil. Parasit., 16, 82. OVEmgULVE, J. P. (1970). Koninkle. Nederl. Akademie van Wettemchappen-Amsterdam Proc. Series C, 73 (1), 129. PEARSE,A. G. E. (1960). Histochemistry TheoreticalandApplied. 2nd Ed., London: J. and A. Churchill. PIEKARSKI, G. (1971). Toxoplasma Colloquium of the Second International Congress of Parasitology and Z. Parasitenk. (In press). SHEFFIELD, H. G.(1970). Proc. helminth. Soc. Wash., 37, 237. - & MELTON, M. L. (1970). Science, N . Y . , 167, 892. SHELTON, G. C. (1968). J. Am. vet. reed. Ass., 152, 1084. , KINTNER, L. D. & MACKINTOSH,D. O. (1968). Ibid., 152, 363. SLIM, J. CHR., HUTCI-IISON,W. M. & WORK, K. (1969). Acta path. microbiol, scand., 77, 756. WAGNER, W.-H. & FOERSTER, O. (1964). Z. Parasitenk., 25, 28. WEILAND, G. & KI3I-IN,D. (1970). Berl. Miinch. Tieriirztl. Wschr., 83, 128. WENYON, C. M. (1923). Ann. trop. Med. Parasit., 17, 231. (1926). Protozoology, A Manual for Medical Men, Veterinarians and Zoologists, Vol. 1. London: Bailli6re, Tindall and Cox. & SHEATHER,L. (1925). Trans. R. Soc. trop. Med. Hyg., 19, 10. WITTE, H. N. & PIEKARSKI,G. (1970). Z. Parasitenk, 33, 358. WORK, K. ~¢ HUTCHISON, W. M. (1969a). Acta path. microbiol, scand., 75, 191. &- (1969b). Ibid., 77, 414.

ADDENDUM Due to the postal strike, several papers have only recently reached us. FRENKEL(1970) reviews the current situation in this field from his point of view and DUBE¥ et al. (1970) describe the o6cyst from cat faeces. ZAMAN & COLLEY(1970) and COLLEYand ZAMAN(1970) give an interesting light and electron microscope account of the endogenous stages of Toxoplasma in the intestinal epithelium of the cat. There seems to be uncertainty as to the actual number o f flagella present in the coccidian microgamete. HAMMONDet al. (1969) state, " A definite conclusion concerning the number of flagella must await further studies" with other species ofcoccidia.