Zeitschrift f~r

Z Parasitenkd (1983)69:447-456 parasitenkunde Parasitology Research

�9 Springer-Verlag t983

Electron Microscope Study of Merogony Preceding Cyst Formation of Sarcocystis sp. in Roe Deer (Capreolus capreolus)

Rolf Entzeroth Abteilung ffir Protozoologie, Zoologisches lnstitut der Universitfit Bonn, Poppelsdorfer Schlog, D-5300 Bonn, Federal Republic of Germany

Abstract. Precystic merogony of Sarcocystis sp. was studied in roe deer fawns 33, 45, and 49 days postinoculation (pi) with 2 x 104 105 sporo- cysts recovered from dogs. Single merozoites, but no meronts, were found 33 days pi in liver, spleen, and lymph nodes. Transforming mero- zoites and meronts were found in myofibroblasts, satellite cells, and endothelial cells of muscle tissue on 45 and 49 days pi; they were sur- rounded by two membranes. Typical coccidian merozoites differentiated simultaneously around an enlarged, lobed nucleus.

Introduction

Species of Sarcocystis are common parasites that infect myocardial and skeletal muscle of many species of mammals, birds, and reptiles (Kalyakin and Zasukhin 1975). Most studies of Sarcocystis spp. have been concerned with the cyst and cyst formation (Mehlhorn et al. 1976). In some species precystic merogony has been associated with pathogenic effects (Fayer and Johnson 1973, 1974; Johnson et al. 1975; Heydorn and Gestrich 1976; Koller et al. 1977). Electron microscope studies of Sarcocystis meronts have revealed that merozoite formation in this genus differs from that of other coccidia (Cernfi and S6naud 1977; Pacheco and Fayer 1977; Heydorn and Mehlhorn 1978; S6naud and Cernfi 1978; Aryeetey et al. 1980; Dubey et al. 1980; Speer and Dubey 1981). The present electron microscope study eluci- dates merozoite development preceding cyst formation in tissues of roe deer experimentally infected with sporocysts from dogs.

Materials and Methods

Oocysts and sporocysts of Sarcocystis sp. were isolated from dogs fed with naturally infected roe deer muscle (esophagus and diaphragm). Feces of two dogs were examined daiIy a week before the beginning of the experiment as welt as during prepatent and patent periods. No coccidian oocysts or sporocysts were detected before day 13 postinoculation (pi). Sporulated

448 R. Entzeroth

oocysts and sporocysts were isolated from the feces either by NaCI floatation or by scraping the mucosa of the small intestine of infected dogs from underlying tissue and treating it with 0.25% trypsin in 0,154 M saline for 20 rain at 37 ~ C. Four roe deer fawns, 3-5 weeks old (three of these were immediately separated from does after birth and raised by hand), two sheep and two mice were inoculated with the following doses of sporocysts: Three roe deer fawns, l0 s each; one roe deer fawn, 2 x 104; two lambs, 5 x 104 and 3 x 10s; two mice, 106 each.

For electron microscope studies, pieces of liver, spleen, mesenteric lymph nodes, tongue, esophagus, and diaphragm were taken from the freshly killed animals (in one case the animal was already dead but still warm) and fixed in 2.5% glutaraldehyde in cacodylate buffer at pH 7.4. Pieces of tissue were postfixed with 2.5 % OsO 4, dehydrated with acetone, and stained with uranyl acetate and phosphotungstic acid in 70% acetone. After final dehydration in acetone, the material was embedded in Vestopal W, sectioned on a Reichert Ultracut micro- tome, and stained with lead citrate. Ultrathin sections were examined in a Zeiss EM 9 $2 electron microscope.

Results

The first roe deer fawn inoculated with 105 sporocysts was killed 33 days pi, shortly before it would presumably have died. Single merozoites, but no meronts, were found in the liver, spleen, and mesenteric lymph nodes; no merogony stages were found in the muscle tissue of this animal. The second roe deer fawn was killed 45 days pi, shortly before it would presum- ably have died. The third roe deer fawn was found dead 49 days pi. The fourth roe deer fawn died 24 h pi of causes other than Sarcocystis infection. Single merozoites and numerous meronts were observed in the muscle tissue of the tongue and esophagus of the second and third animals. However, no parasites were found in the liver and spleen of these animals, nor in sheep (25, 103 days pi) or mice (90 day pi).

Single merozoites were found in myoblasts, satellite cells, and endothelial cells, but not in muscle cells (Figs. 1, 2, 3). They measured 4.4-2.2 (3.2-5 x 1.9-2.3) gm (n=5) in length. The merozoites were bounded by a typical three-membraned pellicle (Figs. 3, 4) consisting of two unit mem- branes, forming the inner membrane complex, and the plasmalemma (Fig. 4). A conoid, micronemes, and a micropore were sometimes visible (Figs. 2, 3). During the transformation of the merozoites into meronts, the three-membraned pellicle disappeared (Fig. 5), and the parasite-host cell contact (PC) zone then included only two membranes (Figs. 5, 6). These two membranes often formed loops and invaginations into the cytoplasm of the meront giving rise to lobes that became closely apposed to the main body of the meront (Fig. 7), resulting in the presence of four adjacent unit membranes (Fig. 8). High-resolution electronmicrographs show that the membrane limiting the parasite (mP) and that limiting the host cytoplasm (mH) were both 6.0-8.0 nm thick. The latter membrane formed by the host cell was clearly trilaminar. The space between parasite and host membranes, 12-14 nm wide, is considered to be a part of the parasitophorous vacuole.

The cytoplasm of the meront was characterized by the presence of mito- chondria with tubular cristae, dense granules, endoplasmic reticulum, and granular cytoplasm (Fig. 5). The nucleus was lobate, the lobes being con-

Figs. 1-4. Sarcocystis sp. from tongue muscle of a roe deer 45 days pi with sporocysts from a dog

Fig. 1. Cross section through the upper apical part of a merozoite with numerous micronemes (MN). The host cell (HC) is a myofibroblast with myofibrils (F) and mitochondria, x 9,450

Fig. 2. Longitudinal section through a merozoite, with a conoid (C), micronemes (MN), and a posteriorly situated nucleus (N). x 9,000

Fig. 3. Longitudinal section through a merozoite with micronemes (MN), mitochondrion (M/) and a nucleus (N) in a host cell. Note the gap in the inner membrane complex of the pellicle (arrow). x 29,250

Fig. 4. Higher magnification of the merozoite pellicle (in the rectangle in Fig. 3) in the area of a micropore (MP) showing the inner membrane complex (IM) and a plasmalemma (OM); adjacent to the parasite pellicle is a membrane of the host cell endoplasmic reticulum (arrow). x 147,600

Figs. 5-8. Sarcoystis sp. from the femoral muscle of a roe deer 49 days pi

Fig. 5. Young meront within a host cell (HC). Note the irregularly shaped nucleus (N) and numerous sections through mitochondria (MI). ER endoplasmic reticulum, x 7,650

Fig. 6. Enlarged area (delimited by the rectangle in Fig. 5) showing the host cell-parasite contact zone consisting of one membrane limiting the parasite's cytoplasm (mP) and another (mH) limiting the cytoplasm of the host cell (Hc). PC host-parasite contact zone. x 38,700

Fig. 7. Section through the peripheral part of a meront with an adjacent cross section of a lobe-shaped invagination in which two limiting unit membranes are seen (rectangle). Note the host cell (HC) with its mitochondria (MIH) and the granular meront cytoplasm (PC) with tubular mitochondria (M/). x 9,000

Microscope Study of Merogony Preceding Cyst Formation of Sarcocystis sp. 451

Fig. 9. Sarcocystis sp. from the esophagus muscle of a roe deer 49 days pi. The meront has a lobate nucleus (N). The forming merozoites (DME) show polar rings (P) and conoids (C). The host cell (HC), with its mitochondria (M/) surrounding the meront, is situated between the sarcoplasm (SP) of adjacent muscle fibers, x 9,900

Fig. 8. Enlarged area (the rectangle in Fig. 7) shows that the cytoplasm of the meronts and its infolded part (PC) is bordered by a unit membrane (mAP). The host cell cytoplasm (HC) is bordered by another unit membrane (mH). The space in between the two membranes is the area of parasitophorous vacuole (PV). M1 mitochondrion, x 126,000

Figs. 10-12. Sarcocystis sp. from the tongue muscle of a roe deer 49 days pi

Fig. 10. Section through merozoites with Golgi complexes (GO), Golgi anlagen (GOA), and inner membrane complex (IM) in the process of differentiation. Note the gaps (arrow) between the plates of the inner membrane complex. The parasite-host cell contact zone forms deep invaginations (IPCO. MI mitochondrion; N nucleus, x 18,900

Fig. 11. Longitudinal section through merozoites (ME) in the final process of differentiation. Note the micronemes (MAr), nuclei (N), mitochondrion, and posterior polar ring (PP). x 13,500

Fig. 12. Enlarged area (the rectangle in Fig. 11) shows the differentiation of the merozoite pellicle. A membrane of the host cell-parasite contact zone becomes attached (arrowheads) to the inner membrane complex (IM) thus forming the merozoite's plasmalemma (ON0. x 24,300

Microscope Study of Merogony Preceding Cyst Formation of Sarcocystis sp. 453

nected by narrow bridges. Beneath and adjacent to the nuclear envelope was an electron-dense layer of chromatin. Meronts measured 15.1-20.0 gm (n--10) in diameter. They varied form spherical to very irregular in shape, with deep invaginations (Fig. 7).

In nearly mature meronts, the nucleus was highly lobate and merozoite primordia formed adjacent to the tips of its lobes (Fig. 9). These primordia consisted of a canopy-like inner membrane complex (IM) plus the future conoid and polar rings. Between each forming apical complex and nuclear lobe there were numerous vesicles of the Golgi complex, micronemes, and forming rhoptries (Figs. 10, 11). Each elongating merozoite remained at- tached to the residual cytoplasm of the meront, while the mitochondrion and nucleus were incorporated into the body. The merozoite then pinched off the residual body near the posterior polar ring (Fig. 11). In the final stage of differentiation, the limiting membrane of the meront became at- tached to the inner membrane complex (IM) of the forming merozoites (Fig. 12), thus forming the merozoite's plasmalemma (OM). The other mem- brane of the former parasite-host cell contact zone disappeared at this stage of development. Fully differentiated merozoites measured 4.07-1.72 (3.2-6.3 x 1.1-2.6) gm (n=10).

Discussion

Two of the four roe deer inoculated with sporocysts of Sarcocystis sp. from dogs were killed; one died during acute sarcosporidiosis. Very patho- genic species of Sarcocystis have been reported previously from cattle, sheep, pigs, and horses (Fayer and Johnson 1974; Gestrich et al. 1974; Munday et al. 1975; Heydorn 1977; Simpson and Mayhew 1980). It is of interest that the lethal phase of sarcosporidiosis in roe deer occurred between 33 and 49 days pi, about 2 weeks later than reported previously in sarcospori- diosis in the domestic animals examined to date. However, the situation observed by us is in agreement with that described for Sarcocystis hemionila- trantis in mule deer by Hudkins and Kistner (1977), i.e., death occurring between 27 and 63 days pi.

We have shown in the present study that merogony is characterized by marked enlargements of the parasite nucleus, formation of lobes, and simultaneous differentiation of many daughter cells in the meront cyto- plasm. Similar observations have been reported from other species of Sarco- cystis (CernA and S~naud 1977; Pacheco and Fayer 1977; Heydorn and Mehlhorn 1978; S~naud and Cernfi 1978; Simpson and Mayhew 1980) and in Frenkelia (G6bel et al. 1978). Cernfi and S6naud (1977) introduced the term" endopolygen~se multiple synchrone" for this phenomenon which they observed in mice after inoculation with S. dispersa.

Of particular interest in the present study was the parasite-host cell relationship during the various stages of infection in different organs. Mero- zoites with a three-membraned pellicle found 33 days pi in spleen and liver cells were situated in the cytoplasm of the host cell without an obvious separating membrane. Similar observations have been reported for S. cruzi,

454 R. Entzeroth

S. suihominis, and S. capracanis (Pacheco and Fayer 1977; Heydorn and Mehlhorn 1978; Aryeetey et al. 1980); they are explainable by the fact that the cells are still motile.

Transforming merozoites and meronts observed 45 and 49 days pi in myoblasts, satellite cells, and endothelial cells, however, were separated from the host cell cytoplasm by a unit membrane. An explanation for this phe- nomenon is that the inner membrane complex of the merozoite disappeared, leaving the merozoite covered by the outer plasmalemma and that the second unit membrane, the limiting membrane of the parasitophorous vacuole, was formed by the host cell. Loss of the inner membrane complex and retention of the plasmalemma occurs routinely during transformation of motile stages into meronts in coccidia, particularly in species of Eimeria (Chobotar et al. 1975). This dedifferentiation process allows the parasite to change from an elongate to a spheroid shape, followed by increase in mass during nuclear division. Cernfi and S6naud (1977) noted interruptions in the inner membrane complex of S. dispersa developing in hepatic cells of mice. Heydorn and Mehlhorn (1978) described all merogony stages of S. suihominis limited by a three-membraned pellicle. Two membranes were observed around meronts of S. cruzi, but these were considered to be part of the parasite (Pacheco and Fayer 1977; Dubey et al. 1980). These authors reported that the host cell cytoplasm was in direct contact with the mem- branes of the parasite. In the present study the parasite-host cell contact zone was seen to consist of two membranes, the plasmalemma of the parasite and the membrane formed by the host cell. The parasite is, therefore, not in direct contact with the host cell cytoplasm, but is separated from the latter by a regular narrow space of the parasitophorous vacuole. A similar narrow space was found between the host cell and developing meronts in some species of Plasmodium (Ladda et al. 1969; Langreth et al. 1978). Fur- ther evidence that the inner membrane (raP) of the parasite-host cell contact zone is the meront plasmalemma and that the outer membrane (mH) is of host cell origin is found during the final stages of differentiation. As the developing merozoite reaches the surface of the meront, the inner mem- brane complex (IM) of the forming merozoite becomes enveloped by the meront plasmalemma (mP) to complete the formation of the pellicle, while the outer host cell membrane disappears. The observed gaps in the inner membrane complex show that this structure is formed by several plates, as has been demonstrated by freeze fracture studies (Dubremetz and Torpier 1978). That the mother cell plasmalemma is incorporated into the merozoite pellicle has also been noted in S. dispersa meronts (S6naud and Cernfi 1978), and it is a common occurence in species of Eimeria (Dubremetz and Elsner 1979; Roberts et al. ] 970; Ferguson et al. 1976). Further studies are needed to clarify if the variations observed in the host cell-parasite contact zone depend on the species characteristics of Sarcocystis or are influenced by the type of host celt involved.

Acknowledgements. This study was supported by a Feodor Lynen stipend from the Alexander yon Humboldt Foundation. I thank Prof. Gulotta and Dr. Htilsmann, Institut ftir Neuropatho-

Microscope Study of Merogony Preceding Cyst Formation of Sarcocystis sp. 455

logie der Universit/it Bonn, for identifying the different cell types, Dr. Ronatd Fayer for reviewing the mansucript, and Prof. E. Scholtyseck for his support of this study.

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Accpeted April 14, 1983