Summary. Intrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was ...
Cell a n d Tissue
Cell Tissue Res (1985) 241 : 353-360
Research 9 Springer-Verlag1985
Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host J.F.G.M. Meis, J.P. Verhave, P.H.K. Jap*, and J.H.E.Th. Meuwissen Department of Medical Parasitology and * Department of Cytology and Histology, University of Nijmegen, Nijmegen, The Netherlands Summary. Intrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite. Key words: Malaria - Plasmodium berghei- Sporozoite Exoerythrocytic form - Liver
The exoerythrocytic (EE) development of a primate malaria parasite in hepatocytes was first described in 1948 using Plasmodium cynomolgi (Shortt and Garnham 1948; Hawking et al. 1948). After the introduction of a suitable rodent model in 1948, it eventually became possible to study the development of P. berghei (Yoeli and Most 1965) in livers of rats. The infection starts when an infected anopheline mosquito takes a bloodmeal from a host and simultaneously injects sporozoites into the bloodstream. In mammalian hosts these highly specialized unicellular parasites find their way to hepatocytes within an hour. Once inside, they reproduce asexually and ultimately form merozoites, after about 50 h in the case of the rodent parasite P. berghei. The merozoites penetrate the red cells and the subsequent asexual cycle of development causes the clinical symptoms of malaria. The latter phase of parasite development has thus far been the most studied part of the plasmodial life cycle (Aikawa 1977; Sinden 1978). The liver forms, which precede the blood infection, have been poorly studied due to their relative inaccessibility. Investigation of the entry and transformation of the invasive sporozoites in hepatocytes in vivo
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presented great difficulties because of the small size of the parasites and their low density in the liver. Using the indirect immunofluorescence assay (IFA) with specific antisera it is possible to identify early EE forms (14-42 h) of P. berghei in fixed liver tissue (Danforth et al. 1978) and in cultured cells (Hollingdale 1983 a, b). Very recently Aikawa et al. (1984) were able to describe briefly the fine structure of early EE forms of P. berghei in vitro. By focusing our efforts on producing high infections in a very small volume of liver in vivo it became possible to follow by electron microscopy the migration of sporozoites from blood to hepatocytes, invasion of hepatocytes and the very early intrahepatocytic transformation (Verhave and Meis 1984). However, although briefly described (Meis et al. 1983c), the early intrahepatocytic growth and the complete transition into the trophic EE form remained a gap in our knowledge of the life cycle of the parasite. In the present study we describe the in vivo intrahepatocytic transformation of the sporozoite of Plasmodium berghei into the young trophic EE tissue stage.
Materials and methods
Laboratory animals Brown Norway rats (BN/BiRij) were obtained from TNO, Rijswijk, The Netherlands. This rat strain was preferred because of its high susceptibility to sporozoites of P. berghei. The following additional measures were taken to reach a high number of infected hepatocytes, which is necessary for ultrastructural studies. (1) The majority of the liver was excluded from the blood stream by temporary ligation of the bloodvessels to the median and left lateral lobes for 60 min (Meis et al. 1981), thereby directing the sporozoites into the small right and caudate lobes (Meis et al. 1981). (2) A volume of 0.5 ml, containing 15-20 x 1 0 6 sporozoites, was injected directly into the portal vein of each rat.
Parasites Sporozoites of P. berghei ( A N K A strain) were harvested from homogenized Anopheles stephensi and separated from mosquito tissue on a biphasic gradient of Urografin| (Schering, Berlin, FRG). To preserve the infectivity of sporozoites the isolation medium contained 10% (v/v) normal rat serum and the suspending medium was kept on crushed
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Fig. 1A-C. IFA of rat liver infected 10 h previous with P. berghei sporozoites. A Transformed sporozoite (arrow) demonstrating a tapered end. Note that fluorescence is mainly associated with the projection and the surface of the bulb. B Another parasite (arrow) with typical surface fluorescence. C Parasite (arrow) with a midregion bulb and two arm-like projections. • 1000 Bar = 10 p.m
ice (Meis et al. 1981). Just before injection the suspension was b r o u g h t to 37 ~ C.
Fig. 2A, B. Toluidine-blue stained semi-thin Epon sections. A EE form of 15 h (arrow) located in the portal region. B Two EE forms of 28 h (arrows). They have completely transformed and are now in the growing phase. • 1000. Bar = 10 p.m
Antiserum T h e a n t i - s p o r o z o i t e s e r u m was p r o d u c e d in rabbits by interr u p t e d biting o f m o s q u i t o e s infected with P. berghei, as p r e v i o u s l y described ( V e r m e u l e n et al. 1982). T h e i m m u n e s e r u m was c h e c k e d for specificity by I F A using purified s p o r o z o i t e s as slide a n t i g e n and fluorescein-labeled swine a n t i - r a b b i t I g G a n t i b o d i e s ( N o r d i c | Tilburg, The N e t h e r lands) as a c o n j u g a t e .
lmmunoJTuorescence Livers o f rats i n o c u l a t e d 10 h p r e v i o u s l y with sporozoites were p e r f u s i o n fixed with 0 . 1 % g l u t a r a l d e h y d e / 2 % p a r a f o r m a l d e h y d e in 0.1 M c a c o d y l a t e buffer, rinsed in repeatedly c h a n g e d buffer for 24 h in a shaker at 4 ~ C, d e h y d r a t e d
in g r a d e d e t h a n o l s and e m b e d d e d in E p o n 812. Semi-thin (0.5 ~tm) sections were cut with glass knives and m o u n t e d on glass slides c o a t e d with c h r o m e a l u m gelatine (0.05 g c h r o m e a l u m plus 0.5 g gelatin in 100 ml distilled water). T h e sections were de-eponized for 1 h in s a t u r a t e d solution o f s o d i u m h y d r o x i d e in a b s o l u t e ethyl a l c o h o l (Lane and E u r o p a 1965) and subsequently r e h y d r a t e d to p h o s p h a t e buffered saline (PBS, p H 7.2). T h e sections were reacted with rabbit a n t i - s p o r o z o i t e s e r u m (titer 1:5120-10,240) for 20 m i n at 37 ~ C, rinsed for 45 min in PBS a n d reacted with swine anti-rabbit i m m u n o g l o b u l i n e s c o n j u g a t e d to fluorescein i s o t h i o c y a n a t e (Nordic| The sections were viewed with a Leitz O r t h o l u x II e q u i p p e d with a Leitz V a r i o - o r t h o mat.
Fig. 3. A An oblique section through the anterior portion of a sporozoite ( ~ 0.8 p.m) of P. berghei found in a hepatocyte one hour post-inoculation (PI). The trilaminar pellicle consisting of an outer plasmalemma and two tightly opposed inner membranes (IM), and the subpellicular microtubules (MT) are apparent. The parasite cytoplasm reveals numerous micronemes (MN) and two osmiophilic bodies (arrows). • 32500. Bar=0.5 ~tm. B A cross-section of a sporozoite ( ~ I ~tm) within the parasitophorous vacuole (PV) of the hepatocyte observed 2 h PI. An osmiophilic membrane-whorl is present in the vacuole (arrow). • 20500. Bar = 0.5 pro. The inset reveals the outer sporozoite plasmalemma, inner membranes (IM) and the underlying microtubules (arrows) x 78000. Bar = 100 nm. C A parasite ( ~ 1.6 p.m) in a hepatocyte 4 h PI. The typical osmiophilic membrane-whorls are still visible in the PV (arrows). Note that the host hepatocyte is binuclear (HN). • 28500. Bar=0.5 p.m. Inset reveals the part of the parasite pellicle with the subpellicular microtubules (arrows). • 9500. Bar= 1 p.m. D Another parasite ( ~ 1.8 p.m) observed 4 h PI. Most of the rigid pellicle is still intact (microtubules, arrows), x 33000. Bar= 1 ~tm. E Parasite found 10 h PI ( ~ 2.7 ~tm). The typical sporozoite pcllicle is clearly visible around the parasite. The nucleoplasm (N) exhibits the same texture as the cytoplasm. Rough endoplasmic reticulum is associated with the nucleus (arrowheads). A large membrane-whorl is apparent (arrow). A glancing section through the tapered end of the parasite demonstrates arrays of parallel running microtubules (MT) SD Space of Disse. • 13 500. Bar = 1 p.m. F Detail of the indicated area in Fig. 3 E showing the longitudinal section through the microtubules (MT) ( ~ ca. 22 nm). Osmiophilic material in the PV is still visible (arrow) x 59500. Bar=100 nm. G Parasite in the process of dedifferentiation ( ~ 3 p.m) observed 15 h PI. Arrows indicate the area of the persisting trilaminar pellicle. The other part of the parasite has only one limiting parasite plasmalemma giving it an irregular outline. The cytoplasm is filled with polyribosomes. The nucleus (N) is characterized by the absence of ribosomes. The typical demarcation (arrowheads) running around the nucleus is a R E R lamella. Several moderately dense membrane-bound vesicles with the same diameter (60-120 nm) as the micronemes of sporozoites are observed (thin arrows). Remnants of osmiophilic bodies (OB) (see Fig. 3A) are visible, x 16000. B a r = l p.m. H Detail of another section from the indicated area of G. The obliquely sectioned remnants of microtubules (MT) are clearly visible. These microtubules are associated with the persisting inner membranes (IM). The single parasite plasmalemma (PP) is shown at the right. Note remnant of osmiophilic body (arrow). • 49500. Bar = 200 nm
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Fig. 4. A Parasite observed 15 h PI. This longitudinal section reveals the tapered end of the partially dedifferentiated sporozoite. The apical end with the polar ring (arrows) and attached microtubules (MT) are clearly visible. The typical pellicle is present over half the surface area of the parasite. The other part has a trophic appearance. The two inner membranes of the parasite have partly pulled away from the outer parasite plasmalemma (thin arrows). Nucleoplasm (N) can be differentiated from the cytoplasm by the absence of ribosomes. No evidence of persisting rhoptries is observed. Note the remnant of the osmiophilic dense bodies (thick arrow). • 21000. Bar = 1 I,tm. B Detail of Fig. 4A. The composition of the pellicular membranes is clearly revealed. The PV membrane (PVM) has the same thickness (ca. 6 nm) as the parasite plasmalemma (PP). The thick layer of two closely opposed inner membranes (IM) and the subpellicular microtubules (MT) run until the apical ring (AR). x49500. B a r = 2 0 0 nm. C Another oblique section through the same anterior end shows the fan shaped arrangements of the microtubules (MT) attached to the apical ring (AR). • 49500. B a r = 200 nm. D Two thirds of this parasite of 19 h (~Z~3.4 ~tm) is still covered by the inner membranes (between arrows) and subpellicular microtubules (MT). Apart from many ribosomes, some mitochondria (M) and dilated RER strands are visible. The nucleus is not sectioned in this parasite. • 23500. B a r = 1 ~tm. E Low power electron micrograph of a small EEF of 21 h ( ~ 4.2 ~tm) located in a binuclear hepatocyte (HN) in the periportal area of the liver. Note the venule (V), bile duct (BD) and sinusoids (S). • 3000. Bar = 5 ~tm
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Light- and electron microscopy To study transformation and development of the early sporozoite, rats were sacrificed at 1, 2, 4, 10, 15, 19, 21, 24 and 28 h after inoculation of sporozoites and the livers were perfused with 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) at room temperature. The livers were sliced and immersed for another 2 h in the same fixative at 4 ~ C, followed by rinsing in buffer for at least 3 h. The tissues were post-fixed in buffered i % osmium tetroxide at 4 ~ C for 2 h and rinsed in buffer for at least I h. Dehydration was performed in an ascending series of aqueous ethanols and the tissues were transferred via a mixture of propylene oxide and Epon to pure Epon 812. Semithin sections were cut from material of 15 h and later, stained with 5% Toluidine blue in 5% borax, and examined for EE forms in the periportal areas (Meis et al. 1983a). Younger parasites (i.e., 1-10 h) are too small to be detected by light microscopy. Therefore, ultrathin sections were immediately cut around afferent portal tracts. The sections were cut with a diamond knife, contrasted with uranyl acetate and lead citrate, and examined in a Philips EM 301 electron microscope. Results
Immunofluorescence and light microscopy Semithin de-eponized liver sections fixed in 0.1% glutaraldehyde/2% paraformaldehyde revealed intracellular parasites of various shapes (diameter about 2.5 [am) after 10 h with the IFA (Fig. 1). They had a comma-shape (Fig. 1 A), were round (Fig. 1 B) or bulb-like with two extensions (Fig. 1 C) and were not visible when control serum was applied. Despite extensive searching no EE forms could be detected in sections stained normally with Toluidine blue in the first 15 h after sporozoite inoculation. From 15 h onwards parasites could be located in semi-thin sections (Fig. 2A, B) and subsequently were sectioned for electron microscopy.
Electron microscopy Although immunofluorescence and light microscopy were useful for localization of the EE forms, electron microscopy was necessary for confirmation of parasitic nature and interactions of the parasite with its host. Sporozoites detected in hepatocytes up to 2 h post-inoculation (PI) with a diameter of 1 [am, revealed a sickle shape and a structural organization which was well conserved (Fig. 3 A, B). The intracellular sporozoites were all localized inside a membrane-bounded parasitophorous vacuole. The parasites had a trilaminar pellicle, beneath which lay an inner layer of subpellicular microtubules (MT) attached to a rigid apical ring at the anterior end. Transformation of the invasive sporozoites into the trophic EE form was accompanied by loss of pellicular rigidity (Fig. 3 C) together with bulbous enlargement around the nucleus. However both at 4 h (Fig. 3C, D) and 10 h (Fig. 3E) PI, we observed most of the trilaminar pellicle with the attached MT still covering a major part of the total surface area. Typical osmiophilic membrane-whorls in the parasitophorous vacuole were visible until 10 h PI (Fig. 3C, D, E, F). The bulbous part of the parasite had an average diameter of 1.5 [am at 4 h (Fig. 3D) and 2.7 [am at 10 h (Fig. 3E). The remainder of the tapered sporozoite tail could be observed
up to 15 h (Figs. 3F, 4A, B, C). At 10 h PI the parasites had become pear-shaped (diameter about 2.7 [am) or spherical and were located near the lateral surface of the host cell (Fig. 3 E). The trilaminar pellicle with attached MT covered three-fourths the total surface. A tangential section through the tapered end that protrudes into the hepatocyte cytoplasm demonstrated the radiating MT (Fig. 3 E, F). At 15 h PI the parasites measured about 3 [am in diameter. The anterior end with its apical complex was tapered from the rest of the parasite (Fig. 4A). The inner membranes and MT persisted over the anterior region and over half of the main cell body. The other half had lost this rigid appearance and assumed atrophic aspect. The inner membranes appeared to be pulled away from the outer plasmalemma (Fig. 4A) and overgrown by parasite cytoplasm. Although the apical ring with attached MT was still visible (Fig. 4 B, C) the distinct rhoptry/microneme complex typical in sporozoites (Fig. 3A) had disappeared. It was possible to observe only some vesicles (60-120 nm) with electrondense material. They were of the same size as micronemes (Fig. 3 G), and therefore might be such. At 19 h PI the parasites were more or less spherical with a diameter of about 3.4 [am. The rigid pellicle occupied up to two thirds of the total surface area (Fig. 4D). Several mitochondria, often cup-shaped, some dilated ER stacks, and a Golgi-complex were visible. At 21 h PI the parasite had expanded to about 4.2 [am in diameter. Parasites were regularly encountered in the periportal area in binuclear hepatocytes (Figs. 3 C, 4 E). The trilaminar pellicle now occupied only half of the total surface area (Fig. 5A). The ER had largely proliferated, exhibiting dilated cisternae. The cytoplasm contained packed ribosomes, many of them as polysomes indicating extensive protein synthesis. At this stage of development, a mitotic spindle was apparent as the first indication of nuclear division (Fig. 5A). At 24 h the EE form had increased in size (average diameter 7.5 lam) still with some persisting inner pellicular membranes (Fig. 5 B). Nuclear divisions were indicated by the large nuclear profiles and mitotic spindles (Fig. 5B, C). The ER had expanded further. Double-membraned, elongated mitochondria were often located just beneath the parasite plasmalemma (Fig. 5 B, C). This was especially apparent in a superficial section of a 28 h EE form (Fig. 5 F). Parasites of that age had further expanded to about 10 [am. But most striking was the observation that parts of the inner pellicular membranes with the attached MT had still persisted (Fig. 5 D, E, F). Projections of the parasite into the hepatocyte cytoplasm were visible still with the inner membranes attached (Fig. 5 E). Discussion
Studies of the elusive early interaction of sporozoites of
P. berghei with host hepatocytes have been made possible by the use of a very susceptible host, the Brown Norway rat (Meis et al. 1983a). Penetration of sporozoites into hepatocytes is effected by the invagination of the outer plasmalemma (Meis et al. 1983b) thus resulting in a parasitophorous vacuole membrane of host origin. This membrane persists in surrounding the sporozoite during its transformation and growth inside the hepatocyte. Initial dedifferentiation results in the sequential loss of the rhoptry-microneme complex, inner membranes and MT (Meis et al. 1983c). Micronemes could be observed until about 15 h PI.
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359 The partial degradation of the inner membranes and MT implicates the destruction of the rigidity and shape of the sporozoite and permits this intracellular parasite to enter its trophic phase. Our present study also presents clear-cut evidence of the existence of a persisting sporozoite pellicle until at least 28 h. It is not observed in sections of EE forms 30 h PI and later (Meis et al. 1981). It is striking that in the present work and in previous immunofluorescence studies of parasites older than 14 h (Danforth et al. 1978), that young EE forms react readily with polyvalent anti-sporozoite antiserum until 30 h. Thereafter they react only with antiserum against the erythrocytic forms. This reaction pattern o f the EE form is the same using a monoclonal antibody raised against a 44 kD protein on the sporozoite surface (Aikawa et al. 1981). Therefore it is highly likely that the change in antigenicity of the EE form is caused by the disappearance of the last remnants of the sporozoite pellicle. The combined effects of the degeneration of several specialized organelles of the sporozoite leaves the young EE form in a dedifferentiated state with only a few essential structures, such as a nucleus, mitochondrion, some E R and ribosomes enclosed by a limiting membrane of the parasite. F r o m our observations it is apparent that during the first 15-20 h, as the parasite grows from 1 to 3 gm, the expansion of the nucleus and proliferation of organelles like E R and mitochondria are relatively limited. This suggests that during this time-phase, primarily dedifferentiation takes place. F r o m 15 h onwards, however, the parasite grows from 3 to 10 gm at 28 h. In this period the ER becomes markedly expanded with dilated cisternae as known from the older stages (Meis et al. 1981). The cytoplasm contains a high density of polyribosomes. The single mitochondrion of the sporozoite has apparently generated daughter ones which are especially located as elongated tubular structures just beneath the plasmalemma. The nucleus enlarges rapidly and becomes lobed due to the accelerated endomitosis. Several mitotic spindles are present and radiate into the nucleoplasm. All of these morphological changes are indicative of an increase in the rates of synthesis of nucleic acid and protein to produce large numbers of merozoites 24 h later. Despite the unique nature o f the different developmental stages in the life cycle of the malaria parasite, the basic mechanism of transformation o f the ookinete into oocyst (Canning and Sinden 1973; Davies 1974) and of the merozoite into erythrocytic trophozoite (Ladda 1969) resembles that of the sporozoite into EE form. Recent fine structural observations by Aikawa et al. (1984) of transformation of P. berghei in human hepatoma cells in vitro show differences with the present work in vivo. They report a disintegration of the parasitophorous membrane after entering of the sporozoite into the host cell. Our present and previous results (Verhave and Meis
1984) clearly show the parasitophorous membrane to be present until late schizogony, which confirms results obtained by I F A studies (Hollingdale et al. 1983a). F r o m the work of Aikawa et al. (1984) it also appeared that the inner membranes of the transformed sporozoite had disappeared before any morphological nuclear changes were visible. On the contrary our results show clearly that the inner membranes and microtubules persist at least until 28 h PI, a time at which already many nuclear profiles were visible. This would mean that the transformation o f P. berghei sporozoites in vitro is different from the in vivo situation. In vitro, P. berghei sporozoites are able to penetrate and grow intranuclearly (Aikawa et al. 1984; Meis et al. 1984b). This was never observed in vivo (Meis et al. 1984b). On the other hand late EE development of P. berghei in vitro appears to be similar, although somewhat slower, as in vivo (Meis et al. 1984a). The disappearance of the apical complex in the ookinete is reported as 'retraction' into the cytoplasm of the young oocyst. We did not observe such a mechanism in the sporozoite, but the pulling away of the inner membranes might be a stage of internalization through cytoplasmic overgrowth. The process of pinching-off of the anterior end with apical complex, as was assumed previously (Meis et al. 1983c), should not be excluded totally. The transition o f the round merozoite into the young trophozoite happens within 10 min (Bannister et al. 1977). Recent studies on the fate of irradiated sporozoites in tissue culture cells suggest that the transformation of sporozoites takes place during their first 24 h of intracellular life as judged by immunofluorescence studies (Ramsey et al. 1982). Thereafter further development appears to be impaired by alteration o f the nuclear apparatus affecting division. This correlates well with earlier assumptions on abortive infections (Verhave 1975) and with the present electronmicroscopic observations in vivo, which show that in this period the parasite has left its transformation phase and entered its growth phase. The hypnozoites of primate Plasmodium cynomolgi, which are reported to be 4~5 gm (Krotoski et al. 1982a, b), are comparable in size to transforming P. berghei of about 20 h. The latter have started their nuclear division by then. The earliest detectable EE form in the Aotus monkey is at 36 h and measures 2.5 gm in diameter (Krotoski et al. 1982b). Although this is equivalent to the rodent parasite at about 10-15 h, the latter are still in the process of dedifferentiation. A direct comparison may not be possible but both the inactive hypnozoite and the early form o f both active and dormant liver forms are likely to have retained a partly intact trilaminar pellicle and MT. The recent success in the cultivation o f the EE form o f the primate parasite P. vivax in human hepatocytes (Mazier et al. 1984)
Fig. 5. A Magnification of Fig. 4E showing that the inner membranes still cover half of the EE form ( ~ 4.2 gm) surface area (between arrows). A nuclear fragment with a spindle (thin arrow), RER, a mitochondrion (M) a clear vacuole (V) and many ribosomes are apparent, x 11400. Bar= 1 gm. B E E form of 24 h ( ~ 7.5 gm) has still a strand of inner membranes (IM) (see inset: magnification of the indicated area). Several nuclear fragments (N), peripherally located mitochondria (M) and large areas of RER are visible. BC bile canaliculus. Inset: x 30900, x 11400. Bar= 1 lam. C Detail of another 24 h parasite showing a large nuclear reticulum (iV) and mitotic spindles (thin arrows). Large areas of RER and peripherally located mitochondria (M) are clearly visible. The persisting inner membranes are visible between the arrows, x 12800. Bar=l gin. D A greatly enlarged EE form of 28 h (~Z~10 gm) however, inner membranes are still present (arrows). Nuclear fragments (N), large areas of RER and peripherally located elongated mitochondria (M) are apparent, x 4900. Bar= 5 gm. E Detail of the host/parasite interface with the persisting inner membranes (IM) visible even in the protrusion into the host (arrow). Few transverse sections through subpellicular microtubules (thin arrows) are obvious, x 36100. Bar = 0.5 gm. F Grazing section through a parasite of 28 h demonstrating the peripheral localization of the mitochondria (34). x 15200. Bar = 1 gm. Inset shows the persisting subpellicular microtubules (arrows) at a higher magnification, x 27100. Bar = 250 nm
360 and of the hypnozoites in h u m a n hepatoma cells (Hollingdale et al. 1985), could initiate ultrastructural studies of hypnozoite formation, which are hardly possible with biopsy material, due to various technical problems. The host hepatocytes do not show major alterations because of the presence of young EE forms. It could be seen regularly that an EE form inhabits a binuclear hepatocyte. This is also reported for other rodent parasites (Boulard et al. 1983) and holds for hypnozoites of P. cynomolgi as well, as seen in the photographs by Krotoski et al. (1982a, b). Moreover, in vitro studies of EE forms of hum a n P. falciparum often show parasites inhabiting host cells with more than one nucleus (Smith et al. 1984; Mazier et al. 1985), which has been recently confirmed by electron microscopy (Meis et al. unpublished results). In addition microtubules persisted for at least 7 days in growing EE forms of P. falciparum in vitro.
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