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Fine structure of Sarcocystis arieticanis Heydorn, 1985 in its intermediate and final hosts (sheep and dog)
Zbl. Bakt. Hyg. A 264, 353-362 (1987)
Fine Structure of Sarcocystis arieticanis Heydorn, 1985 in its Intermediate and Final Hosts (Sheep and Dog)~' ALFRED OTTO HEYDORN 1 and HEINZ MEHLHORN 2 1
2
Institut fur Parasitologie und Tropenveterinarmedizin, FU Berlin, 1000 Berlin Institut fur Spezielle Zoologie und Parasitologie, Ruhr-Universitat Bochum, 4630 Bochum, Federal Republic of Germany
With 13 Figures' Received March 20, 1986
Summary The developmental stages of S. arieticanis (sheep - dog - cycle) were studied by means of electron microscopy. After oral infection of the sheep with sporocysts two generations of schizonts occur around days 14 and 27 p. i. Both are morphologically more or less similar and are situated immediately inside the cytoplasm of their host cells, which are endothelial cells. They produce somewhat more than 100 merozoites reaching a size of about 6-8 f.lm x 2.5 f.lm. The tissue cysts are characterized by hair-like protrusions of the primary cyst wall. These protrusions (reaching a length of 11 f.lm at the maximum) do not contain fibrils, are folded over and thus running along the surface. These cysts may then appear in light microscopical sections with a smooth cyst wall. The fine structure of the cyst wall was significantly different from the other sheep sarcosporidia. No differences were found in morphology of the cyst merozoites reaching a size of 15 x 2.5 f.lm. Inside the dog gamogony occurred in the cells of the lamina propria of the lower part of the small intestine. Gamogony was mostly completed after 20 h p. i. and sporogony after 12-14 days p. i. The fine structure of the stages in the final host was similar to that of other Sarcocystis species.
Introduction The life cycles of numerous Sarcocystis species have been described in recent years (Lit. c. f. Mehlhorn et aI., 1986). These life cycles always involve two vertebrate hosts: a "prey" (i. e. herbivores, omnivores) and a "predator" (i. e. carnivorous animals, man), with schizogony taking place in the "prey" (= intermediate host) and sexual reproduction of the parasite restricted to the small intestine of the "predator" (= final host). Originally it was thought that each animal species was parasitized as intermediate host by only one sarcosporidian species and that the different cysts seen within the muscles were developmental stages of one cyst type. However, by transmission experi'f Presented at the 5th German-Japanese Cooperative Symposium on Protozoan Diseases, Tokyo, Japan, Sept. 25-28, 1985. Supported by DFG.
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ments (Lit. c. f. Heydorn et al., 1975; Mehlhorn and Heydorn,1978; Frenkel et aI., 1979) was shown that final hosts as well as intermediate hosts may be parasitized by several Sarcocystis species. This needed in our opinion a new nomenclatural definition of these Sarcocystis species (Heydorn et al., 1975; Frenkel et al., 1979, 1980, 1984; Mehlhorn et al., 1985). This absolute need of a new species description was recently underlined by the finding that even the same host range (dog-goat) may be used by biologically and morphologically different Sarcocystis species, which were described as S. capracanis (Fischer, 1979; Heydorn and Haralambidis, 1982) and S. hircicanis (Heydorn and Unterholzner, 1983). Recent transmission experiments (Heydorn, 1985) elucidated that also two Sarcocystis species of sheep use the dog as final host. The first was described as S. ovicanis (Heydorn and Gestrich, 1976), whereas the other was named S. arieticanis (Heydorn, 1985). With respect to their appearance they are both significantly different from the third (cat-transmitted) species: S. ovifelis (Heydorn et al., 1975). The present study deals with the fine structure of the new species S. arieticanis in its both hosts, since fine structure was used as additional.species marker (Mehlhorn et al., 1985).
Materials and Methods The experimental design was already given in a previous paper (Heydorn, 1985). For light and electron microscope studies infected lambs were killed on days 14,26,27,42,56, 70 or 105 p. i.; dogs 15 h, 20 h, 10, 14-16 days p. i. Pieces of the mesenterium and various organs were taken from lambs killed on days 14,26 and 27 p. i., whereas small portions (2 X 2 x 3 mm) of muscles were taken from the other killed lambs. For studies of the parasitic stages of the dog, pieces of the mucosa of the ileum and of the last 30-40 em of the jejunum were taken. All material was then placed in a container of 5.0% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and fixed for 24 h at 4°C in a refrigerator. After rinsing in the buffer the specimens were treated for 2 h in 2 % (w/v) OS04 and dehydrated in an ethanol series before being embedded in Araldite (Ciba-Geigy). Ultrathin sections of the embedded material were cut on a Reichert u!tratome, mounted on copper grids, stained with an alcoholic solution of uranyl acetate for 30 min, and finally in lead citrate for 10 min. Ultrathin sections were then examined in a Zeiss electron microscope EM 9 S 2.
Results
1. Schizonts The fine structure of the first and second generation schizonts, which were seen in endothelial cells on days 14 p. i. and 26 p. i., respectively was nearly identical. Those of the first generation occurred mainly in the arteries of the mesenterium and mesenterial lymph nodes, whereas the second generation schizonts were seen mainly in capillaries of various organs. They reached a size of 90 X 50 flm at the maximum (Figs. 2, 3, 6) and were situated immediately inside the cytoplasm of their host cell; a parasitophorous vacuole was never formed (Fig. 1; He). The surface of the schizonts was bordered by two membranes (Fig. 1; LM) and became constantly enlarged by deep invaginations (Figs. 1,2; IN) leading to aspects of compartments. The homogeneous cytoplasm of the spherical to ovoid schizont became filled by a large polymorphous nucleus, which divided constantly by constrictions giving rise to about 30-70 nuclear portions (Fig. 3; N). During the last division of the latter (Fig. 4; N) the anlagen of the merozoites (Fig.
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Beginning from the 42nd day p. i. tissue cysts were observed inside the muscle fibers. These cysts were bounded by a typical sarcosporidian primary cyst wall (Fig. 8; PW) consisting of a unit membrane with underlying electron dense material at most places. The primary cyst wall formed in mature cysts flattened hair-like protrusions, which were folded over, so that sections in light microscopy revealed a smooth cyst wall. In electron microscopy mainly cross or transversally sectioned protrusions were observed giving rise to a layer of about 1-3 !lm in diameter (Fig. 7; PT). The individual length of each protrusion was only measured clearly at the tips of the cyst, where they reached about 11 !lm in length and a diameter of 0.8 !lm at the maximum. However, these protrusions were shorter in the midregions of the cysts. Cysts found on days 42 and 56 contained spherical to ovoid metrocytes with a longest diameter of about 10-14 !lm (Fig. 7; MC). These metrocytes were bounded by a pellicle consisting of two membanes and provided with typical micropores. In general the metrocytes had a relatively electron-pale cytoplasm with only a few of the significant organelles. However, they had a conoid and reproduced by repeated endodyogenies (Fig. 7); MC, DC). During these endodyogenies the newly formed daughter cells achieved more and more the shape and characteristic features of the finally differentiated (= infectious) cyst merozoites (Figs.
5,9). The mature cyst merozoites measured about 15 !lm X 3 !lm and showed all characteristics of motile infectious stages (sporozoites, merozoites) of sporozoa such as a threelayered pellicle, rhoptries, micronemes, conoid, 22 subpellicular microtubules, polysaccharide granules etc. (Figs. 5, 9). These cyst merozoites were always found in chamberlike hollows of the fine ground-substance (as in Fig. 7; GS). Although cysts on day 105 p. i. were predominantly filled with cyst merozoites, they also contained some metrocytes. These metrocytes were mostly situated at the poles of the cysts and at their periphery; only a few were found in the center of the cysts. These findings indicate, that cysts were still able to grow to some degree, even when they were already macroscopically visible. A secondary cyst wall was never formed around a parasitized muscle fibre, even not in those cysts, which were macroscopically visible. The parasitized host cell remained recognizable as a muscle fiber and was not structurally changed. On the other hand cyst destruction by host defense cells was found to begin on day 70 p. i. in some places and increased till day 105 p. i. 2. Tissue cysts 4; MA) were formed above each tip of the dividing nucleus. At this stage of development the conoid, the inner membranes of the pellicle, a mitochondrion and a few micronemes became visible (Figs. 3, 4). When protruding these anlagen gave rise to probably more than 100 of the finally differentiated merozoites (Fig. 6). These motile stages measured about 6-8 !lm x 2.5 !lm and showed the typical organelles of merozoites as pellicle, conoid. micronemes, rhoptries, golgi apparatus and a long tubular mitochondrion. Furthermore they were characterized by 22 subpellicular microtubules and by the absence (or scarce occurrence) of rhoptries and polysaccharide granules (Fig. 6). When these merozoites were differentiated, the cytoplasm of the host cell was mostly degenerated, and by bursting of its remnants the merozoites were set free and became distributed via the blood stream.
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3. Stages in the dog In sections of the intestine taken 15 h p. i. the cells of the lamina propria contained mainly stages in syngamy (Figs. 10, 11), whereas only a few macrogametes and microgametes were seen. Both types of the gametes corresponded to those seen in other Sarcocystis species (Mehlhorn and Heydorn, 1979). Twenty hours p. i. mainly young oocysts were found during formation of the oocyst wall by fusion of dense inclusions at the periphery of the zygote (Fig. 12). On day 10 p. i. exclusively oocysts were observed inside the cells of the lamina propria. Most of these oocysts contained two sporocysts measuring about 16 X 9 !-tm, however, sporozoites were not yet present (Fig. 13). The oocyst wall was relatively smooth and appeared partly broken in ultrathin sections of still intracellularly situated oocysts. The host cell was degenerated at this time and appeared as an electron dense layer (Fig. 13; RH).
Discussion S. arieticanis Heydorn, 1985 is besides S. ovicanis Heydorn and Gestrich, 1976 the second Sarcocystis species, which has dogs as final and sheep as intermediate hosts. In addition to these two species a third Sarcocystis species is known (up to now) from sheep and called S. ovifelis Heydorn et al., 1975, since it is transmitted by cats. With respect to morphology there is no great difference in schizogony of sarcosporidia. However, cyst morphology and cyst fine structure allow in many cases a signjficant species determination. In the present study it was shown that the cyst wall of S. arieticanis cysts has long, flattened hair-like protrusions, which are folded over, so that in light microscopy cyst walls might appear smooth. In S. ovicanis, however, the protrusions are palisade-like (without fibrils), reach a length of about 3.5 !-tm at the maximum, but stand upright and thus the cysts appear in light microscopy with a thick cyst wall (Mehlhorn et al., 1975). The cysts of S. ouirelis are completely different from these both types. The protrusions are cauliflower-like, reach a length of 4.5 !-tm and contain numerous filaments (Senaud, 1967; Mehlhorn and Scholtyseck, 1973). In addition to these differences of the primary cyst wall, S. ovifelis has a secondary cyst
Figs. 1-13. Sarcocystic arieticanis. Transmission A Amylopectin granule = Conoid C CM = Cyst merozoite = Daughter cell DC DO Developing oocyst wall EN Endothelial cell GS = Ground substance = Host cell HC IC = Interior cavity of the capillary IN = Infolding of the schizont's surface LM - Limiting membranes MA Merozoite anlage MC - Metrocyte MG - Macrogamete MI = Mitochondrion
electron micrographs. ~ MIG = Microgamete MN = Micronemes N = Nucleus NH = Nucleus of the host cell P = Protrusions of PW PV = Parastiophorous vacuole PW = Primary cyst wall R = Rhoptrie RH = Remnant of host cell SC = Schizont ST = Subpellicular microtubules SW = Sporocyst wall TH = Thin oocyst wall V = Vacuole
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e Figs. 1-2. Parasitic stages in the sheep. 1/2. Sections through a developing second generation schizont inside an endothelial cell. Note that the schizont is situated immediately inside the cytoplasm of the host cell and that its surface has deep invaginations (Fig. 1; IN). The nuclei divide constantly by fissions. 1. X 31,500; 2. X 3,500.
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Figs. 3-5. Parasitic stages in the sheep. 3/4. Schizonts during the phase of daughter cell formation. Note that above the two tips of a dividing nucleus always a merozoite analge is formed. Fig. 4 is a magnification of a part of Fig. 3 (No.4). 3. x 4,130; 4. x 17,500. 5. Section through differentiated cyst-merozoites. Note that 22 subpellicular microtubules are present (like in merozoites formed by schizonts). x 28,000.
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Figs. 6-9. Parasitic stages in sheep. 6. Section through a second generation schizont with differentiated merozoites (ME). X 6,175. 7. Section through a young muscle-cyst containing metrocytes (MC), which form daughter cells (DC) by endodyogeny. x 6,500. 8. Section through the primary cyst wall (PW), which forms flattened protrusions. X 16,250. 9. Longitudinal section through a cyst-merozoite (compare Fig. 5). x 6,500. 24 Zbl. Bakt. Hyg. A 264/3--4
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Figs. 10-13. Parasitic stages in the intestinal cells of the dog. 10. Macrogamete with a microgamete in close contact. x 9,100. 11. Microgamete entering a macrogamete. X 27,300. 12. Young oocyst forming the oocyst wall. x 3,500. 13. Sporulating oocyst containing already two sporocysts. X 22,400.
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wall, which is always lacking in S. ovicanis and S. arieticanis. With respect to the morphological findings these three Sarcocystis species of sheep are clearly distinguishable as they are when comparing their biological data (see Heydorn, 1985). S. arieticanis and S. ovicanis represent the second case, where two Sarcocystis species use the same host range. In the other case S. hircicanis/S. capracanis (dog-goat-cycle) also a significant morphological difference of the primary cyst wall led to the final prove of two different species by transmission experiments (Aryeetey et al., 1980; Heydorn and Haralambidis, 1982; Heydorn and Unterholzner, 1983). The morphology of developmental stages of the Sarcocystis species inside their final hosts or in tissue cultures appears very uniform (Mehlhorn and Scholtyseck, 1974; Mehlhorn and Heydorn, 1979; Enzeroth, 1982, present study) and thus give no possibility for species differentiation, however, is characteristic for the genus. On the other hand species may sometimes be differentiated in their final hosts, when their biological data were controlled carefully.
References
Aryeetey, M., H. Mehlhorn, and A. G. Heydorn: Electron microscopic studies on the development of Sarcocystis capracanis in experimentally infected goats. Zbl. Bakt. Hyg., I. Abt. Orig. A 247 (1980) 543-556
Enzeroth, R.: Ultrastructure of gamonts and gametes and fertilization of Sarcocystis sp. from roe deer. Z. Parasitenkd. 67 (1982) 147-153
Fischer, G.: Die Entwicklung von Sarcocystis capracanis n. spec in der Ziege. Vet.-Med. Diss., Bedin FU (1979)
Frenkel, ]. K., A. G. Heydorn, H Mehlhorn, and M. Rommel: Sarcocystinae: Nomina dubia and available names. Z. Parasitenkd. 58 (1979) 115-139
Frenkel, ]. K., A. G. Heydorn, H Mehlhorn, and M. Rommel: Clear communication or arbitrary ambiguity. Z. Parasitenkd. 62 (1980) 199-200
Frenkel,]. K., H Mehlhorn, and A. G. Heydorn: Protozoan Nomina dubia: To Arbitrarily or Replace. The case of Sarcocystis spp.]. Parasit. 70 (1984) 813-815 Heydorn, A. G.: Zur Entwicklung von Sarcocystis arieticanis n. sp. Bed. Munch. tieriirztl. Wschr. 98 (1985) 231-241
Heydorn, A. G., R. Gestrich, H Mehlhorn, and M. Rommel: Proposal for a New Nomenclature of the Sarcosporidia. Z. Parasitenkd. 48 (1975) 73-82
Heydorn, A. G. und R. Gestrich: Beitriige zum Lebenszyklus der Sarkosporidien. VII. Entwicklungsstadien von Sarcocystis ovicanis im Schaf. Bed. Munch. tieriirztl. Wschr. 89 (1976) 1-5
Heydorn, A. G. und S. Haralambidis: Zur Entwicklung von Sarcocystis capracanis Fischer 1979. Bed. Munch. tieriirztl. Wschr. 95 (1982) 265-271
Heydorn, A. G. und]. Unterholzner: Zur Entwicklung von Sarcocystis hircicanis n. sp. Bed. Munch. tierarztl. Wschr. 96 (1983) 275-282
Mehlhorn, H. and A. G. Heydorn: The Sarcosporidia (Protozoa, Sporozoa): Life cycle and fine structure. Advanc Parasit. 16 (1978) 43-72
Mehlhorn, H. and A. G. Heydorn: Electron microscopical study on gamogony of Sarcocystis suihominis in human tissue cultures. Z. Parasitenkd. 58 (1979) 97-113 Mehlhorn, H., A. G. Heydorn,]. K. Frenkel, and E. Gobel: Announcement of the establishment of neohepantotypes for some important Sarcocystis species. Z. Parasitenkd. 71 (1985) 689-692
Mehlhorn, H, D. Diiwel und W. Raether: Diagnose und Therapie der Parasiten von Haus-, Nutz- und Heimtieren. G. Fischer, Stuttgart (1986)
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Mehlhorn, H. und E. Scholtyseck: Elektronenmikroskopische Untesuchungen an Cystenstadien von Sarcocystis tenella aus der Oesophagusmuskulatur des Schafes. Z. Parasitenkd. 41 (1973) 291-310 Mehlhorn, H. und E. Scholtyseck: Licht- und elektronenmikroskopische Untersuchungen an Entwicklungsstadien von Sarcocystis tenelfa aus der Darmwand der Hauskatze. I. Die Oocysten und Sporocysten. Z. Parasitenkd. 43 (1974) 251-270
Senaud, ].: Contribution it l'etude des sarcosporidies et des toxoplasmes. Protistologica 3 (1967) 167-232 Dr. Alfred Otto Heydorn, Inst. f. Parasitologie und Tropenveteriniirmedizin, FU Berlin, K6nigsweg 65, D-1000 Berlin 37
Fine Structure of Sarcocystis arieticanis Heydorn, 1985 in its Intermediate and Final Hosts (Sheep and Dog)~' ALFRED OTTO HEYDORN 1 and HEINZ MEHLHORN 2 1
2
Institut fur Parasitologie und Tropenveterinarmedizin, FU Berlin, 1000 Berlin Institut fur Spezielle Zoologie und Parasitologie, Ruhr-Universitat Bochum, 4630 Bochum, Federal Republic of Germany
With 13 Figures' Received March 20, 1986
Summary The developmental stages of S. arieticanis (sheep - dog - cycle) were studied by means of electron microscopy. After oral infection of the sheep with sporocysts two generations of schizonts occur around days 14 and 27 p. i. Both are morphologically more or less similar and are situated immediately inside the cytoplasm of their host cells, which are endothelial cells. They produce somewhat more than 100 merozoites reaching a size of about 6-8 f.lm x 2.5 f.lm. The tissue cysts are characterized by hair-like protrusions of the primary cyst wall. These protrusions (reaching a length of 11 f.lm at the maximum) do not contain fibrils, are folded over and thus running along the surface. These cysts may then appear in light microscopical sections with a smooth cyst wall. The fine structure of the cyst wall was significantly different from the other sheep sarcosporidia. No differences were found in morphology of the cyst merozoites reaching a size of 15 x 2.5 f.lm. Inside the dog gamogony occurred in the cells of the lamina propria of the lower part of the small intestine. Gamogony was mostly completed after 20 h p. i. and sporogony after 12-14 days p. i. The fine structure of the stages in the final host was similar to that of other Sarcocystis species.
Introduction The life cycles of numerous Sarcocystis species have been described in recent years (Lit. c. f. Mehlhorn et aI., 1986). These life cycles always involve two vertebrate hosts: a "prey" (i. e. herbivores, omnivores) and a "predator" (i. e. carnivorous animals, man), with schizogony taking place in the "prey" (= intermediate host) and sexual reproduction of the parasite restricted to the small intestine of the "predator" (= final host). Originally it was thought that each animal species was parasitized as intermediate host by only one sarcosporidian species and that the different cysts seen within the muscles were developmental stages of one cyst type. However, by transmission experi'f Presented at the 5th German-Japanese Cooperative Symposium on Protozoan Diseases, Tokyo, Japan, Sept. 25-28, 1985. Supported by DFG.
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ments (Lit. c. f. Heydorn et al., 1975; Mehlhorn and Heydorn,1978; Frenkel et aI., 1979) was shown that final hosts as well as intermediate hosts may be parasitized by several Sarcocystis species. This needed in our opinion a new nomenclatural definition of these Sarcocystis species (Heydorn et al., 1975; Frenkel et al., 1979, 1980, 1984; Mehlhorn et al., 1985). This absolute need of a new species description was recently underlined by the finding that even the same host range (dog-goat) may be used by biologically and morphologically different Sarcocystis species, which were described as S. capracanis (Fischer, 1979; Heydorn and Haralambidis, 1982) and S. hircicanis (Heydorn and Unterholzner, 1983). Recent transmission experiments (Heydorn, 1985) elucidated that also two Sarcocystis species of sheep use the dog as final host. The first was described as S. ovicanis (Heydorn and Gestrich, 1976), whereas the other was named S. arieticanis (Heydorn, 1985). With respect to their appearance they are both significantly different from the third (cat-transmitted) species: S. ovifelis (Heydorn et al., 1975). The present study deals with the fine structure of the new species S. arieticanis in its both hosts, since fine structure was used as additional.species marker (Mehlhorn et al., 1985).
Materials and Methods The experimental design was already given in a previous paper (Heydorn, 1985). For light and electron microscope studies infected lambs were killed on days 14,26,27,42,56, 70 or 105 p. i.; dogs 15 h, 20 h, 10, 14-16 days p. i. Pieces of the mesenterium and various organs were taken from lambs killed on days 14,26 and 27 p. i., whereas small portions (2 X 2 x 3 mm) of muscles were taken from the other killed lambs. For studies of the parasitic stages of the dog, pieces of the mucosa of the ileum and of the last 30-40 em of the jejunum were taken. All material was then placed in a container of 5.0% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and fixed for 24 h at 4°C in a refrigerator. After rinsing in the buffer the specimens were treated for 2 h in 2 % (w/v) OS04 and dehydrated in an ethanol series before being embedded in Araldite (Ciba-Geigy). Ultrathin sections of the embedded material were cut on a Reichert u!tratome, mounted on copper grids, stained with an alcoholic solution of uranyl acetate for 30 min, and finally in lead citrate for 10 min. Ultrathin sections were then examined in a Zeiss electron microscope EM 9 S 2.
Results
1. Schizonts The fine structure of the first and second generation schizonts, which were seen in endothelial cells on days 14 p. i. and 26 p. i., respectively was nearly identical. Those of the first generation occurred mainly in the arteries of the mesenterium and mesenterial lymph nodes, whereas the second generation schizonts were seen mainly in capillaries of various organs. They reached a size of 90 X 50 flm at the maximum (Figs. 2, 3, 6) and were situated immediately inside the cytoplasm of their host cell; a parasitophorous vacuole was never formed (Fig. 1; He). The surface of the schizonts was bordered by two membranes (Fig. 1; LM) and became constantly enlarged by deep invaginations (Figs. 1,2; IN) leading to aspects of compartments. The homogeneous cytoplasm of the spherical to ovoid schizont became filled by a large polymorphous nucleus, which divided constantly by constrictions giving rise to about 30-70 nuclear portions (Fig. 3; N). During the last division of the latter (Fig. 4; N) the anlagen of the merozoites (Fig.
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Beginning from the 42nd day p. i. tissue cysts were observed inside the muscle fibers. These cysts were bounded by a typical sarcosporidian primary cyst wall (Fig. 8; PW) consisting of a unit membrane with underlying electron dense material at most places. The primary cyst wall formed in mature cysts flattened hair-like protrusions, which were folded over, so that sections in light microscopy revealed a smooth cyst wall. In electron microscopy mainly cross or transversally sectioned protrusions were observed giving rise to a layer of about 1-3 !lm in diameter (Fig. 7; PT). The individual length of each protrusion was only measured clearly at the tips of the cyst, where they reached about 11 !lm in length and a diameter of 0.8 !lm at the maximum. However, these protrusions were shorter in the midregions of the cysts. Cysts found on days 42 and 56 contained spherical to ovoid metrocytes with a longest diameter of about 10-14 !lm (Fig. 7; MC). These metrocytes were bounded by a pellicle consisting of two membanes and provided with typical micropores. In general the metrocytes had a relatively electron-pale cytoplasm with only a few of the significant organelles. However, they had a conoid and reproduced by repeated endodyogenies (Fig. 7); MC, DC). During these endodyogenies the newly formed daughter cells achieved more and more the shape and characteristic features of the finally differentiated (= infectious) cyst merozoites (Figs.
5,9). The mature cyst merozoites measured about 15 !lm X 3 !lm and showed all characteristics of motile infectious stages (sporozoites, merozoites) of sporozoa such as a threelayered pellicle, rhoptries, micronemes, conoid, 22 subpellicular microtubules, polysaccharide granules etc. (Figs. 5, 9). These cyst merozoites were always found in chamberlike hollows of the fine ground-substance (as in Fig. 7; GS). Although cysts on day 105 p. i. were predominantly filled with cyst merozoites, they also contained some metrocytes. These metrocytes were mostly situated at the poles of the cysts and at their periphery; only a few were found in the center of the cysts. These findings indicate, that cysts were still able to grow to some degree, even when they were already macroscopically visible. A secondary cyst wall was never formed around a parasitized muscle fibre, even not in those cysts, which were macroscopically visible. The parasitized host cell remained recognizable as a muscle fiber and was not structurally changed. On the other hand cyst destruction by host defense cells was found to begin on day 70 p. i. in some places and increased till day 105 p. i. 2. Tissue cysts 4; MA) were formed above each tip of the dividing nucleus. At this stage of development the conoid, the inner membranes of the pellicle, a mitochondrion and a few micronemes became visible (Figs. 3, 4). When protruding these anlagen gave rise to probably more than 100 of the finally differentiated merozoites (Fig. 6). These motile stages measured about 6-8 !lm x 2.5 !lm and showed the typical organelles of merozoites as pellicle, conoid. micronemes, rhoptries, golgi apparatus and a long tubular mitochondrion. Furthermore they were characterized by 22 subpellicular microtubules and by the absence (or scarce occurrence) of rhoptries and polysaccharide granules (Fig. 6). When these merozoites were differentiated, the cytoplasm of the host cell was mostly degenerated, and by bursting of its remnants the merozoites were set free and became distributed via the blood stream.
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3. Stages in the dog In sections of the intestine taken 15 h p. i. the cells of the lamina propria contained mainly stages in syngamy (Figs. 10, 11), whereas only a few macrogametes and microgametes were seen. Both types of the gametes corresponded to those seen in other Sarcocystis species (Mehlhorn and Heydorn, 1979). Twenty hours p. i. mainly young oocysts were found during formation of the oocyst wall by fusion of dense inclusions at the periphery of the zygote (Fig. 12). On day 10 p. i. exclusively oocysts were observed inside the cells of the lamina propria. Most of these oocysts contained two sporocysts measuring about 16 X 9 !-tm, however, sporozoites were not yet present (Fig. 13). The oocyst wall was relatively smooth and appeared partly broken in ultrathin sections of still intracellularly situated oocysts. The host cell was degenerated at this time and appeared as an electron dense layer (Fig. 13; RH).
Discussion S. arieticanis Heydorn, 1985 is besides S. ovicanis Heydorn and Gestrich, 1976 the second Sarcocystis species, which has dogs as final and sheep as intermediate hosts. In addition to these two species a third Sarcocystis species is known (up to now) from sheep and called S. ovifelis Heydorn et al., 1975, since it is transmitted by cats. With respect to morphology there is no great difference in schizogony of sarcosporidia. However, cyst morphology and cyst fine structure allow in many cases a signjficant species determination. In the present study it was shown that the cyst wall of S. arieticanis cysts has long, flattened hair-like protrusions, which are folded over, so that in light microscopy cyst walls might appear smooth. In S. ovicanis, however, the protrusions are palisade-like (without fibrils), reach a length of about 3.5 !-tm at the maximum, but stand upright and thus the cysts appear in light microscopy with a thick cyst wall (Mehlhorn et al., 1975). The cysts of S. ouirelis are completely different from these both types. The protrusions are cauliflower-like, reach a length of 4.5 !-tm and contain numerous filaments (Senaud, 1967; Mehlhorn and Scholtyseck, 1973). In addition to these differences of the primary cyst wall, S. ovifelis has a secondary cyst
Figs. 1-13. Sarcocystic arieticanis. Transmission A Amylopectin granule = Conoid C CM = Cyst merozoite = Daughter cell DC DO Developing oocyst wall EN Endothelial cell GS = Ground substance = Host cell HC IC = Interior cavity of the capillary IN = Infolding of the schizont's surface LM - Limiting membranes MA Merozoite anlage MC - Metrocyte MG - Macrogamete MI = Mitochondrion
electron micrographs. ~ MIG = Microgamete MN = Micronemes N = Nucleus NH = Nucleus of the host cell P = Protrusions of PW PV = Parastiophorous vacuole PW = Primary cyst wall R = Rhoptrie RH = Remnant of host cell SC = Schizont ST = Subpellicular microtubules SW = Sporocyst wall TH = Thin oocyst wall V = Vacuole
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e Figs. 1-2. Parasitic stages in the sheep. 1/2. Sections through a developing second generation schizont inside an endothelial cell. Note that the schizont is situated immediately inside the cytoplasm of the host cell and that its surface has deep invaginations (Fig. 1; IN). The nuclei divide constantly by fissions. 1. X 31,500; 2. X 3,500.
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Figs. 3-5. Parasitic stages in the sheep. 3/4. Schizonts during the phase of daughter cell formation. Note that above the two tips of a dividing nucleus always a merozoite analge is formed. Fig. 4 is a magnification of a part of Fig. 3 (No.4). 3. x 4,130; 4. x 17,500. 5. Section through differentiated cyst-merozoites. Note that 22 subpellicular microtubules are present (like in merozoites formed by schizonts). x 28,000.
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Figs. 6-9. Parasitic stages in sheep. 6. Section through a second generation schizont with differentiated merozoites (ME). X 6,175. 7. Section through a young muscle-cyst containing metrocytes (MC), which form daughter cells (DC) by endodyogeny. x 6,500. 8. Section through the primary cyst wall (PW), which forms flattened protrusions. X 16,250. 9. Longitudinal section through a cyst-merozoite (compare Fig. 5). x 6,500. 24 Zbl. Bakt. Hyg. A 264/3--4
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Figs. 10-13. Parasitic stages in the intestinal cells of the dog. 10. Macrogamete with a microgamete in close contact. x 9,100. 11. Microgamete entering a macrogamete. X 27,300. 12. Young oocyst forming the oocyst wall. x 3,500. 13. Sporulating oocyst containing already two sporocysts. X 22,400.
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wall, which is always lacking in S. ovicanis and S. arieticanis. With respect to the morphological findings these three Sarcocystis species of sheep are clearly distinguishable as they are when comparing their biological data (see Heydorn, 1985). S. arieticanis and S. ovicanis represent the second case, where two Sarcocystis species use the same host range. In the other case S. hircicanis/S. capracanis (dog-goat-cycle) also a significant morphological difference of the primary cyst wall led to the final prove of two different species by transmission experiments (Aryeetey et al., 1980; Heydorn and Haralambidis, 1982; Heydorn and Unterholzner, 1983). The morphology of developmental stages of the Sarcocystis species inside their final hosts or in tissue cultures appears very uniform (Mehlhorn and Scholtyseck, 1974; Mehlhorn and Heydorn, 1979; Enzeroth, 1982, present study) and thus give no possibility for species differentiation, however, is characteristic for the genus. On the other hand species may sometimes be differentiated in their final hosts, when their biological data were controlled carefully.
References
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Enzeroth, R.: Ultrastructure of gamonts and gametes and fertilization of Sarcocystis sp. from roe deer. Z. Parasitenkd. 67 (1982) 147-153
Fischer, G.: Die Entwicklung von Sarcocystis capracanis n. spec in der Ziege. Vet.-Med. Diss., Bedin FU (1979)
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Frenkel,]. K., H Mehlhorn, and A. G. Heydorn: Protozoan Nomina dubia: To Arbitrarily or Replace. The case of Sarcocystis spp.]. Parasit. 70 (1984) 813-815 Heydorn, A. G.: Zur Entwicklung von Sarcocystis arieticanis n. sp. Bed. Munch. tieriirztl. Wschr. 98 (1985) 231-241
Heydorn, A. G., R. Gestrich, H Mehlhorn, and M. Rommel: Proposal for a New Nomenclature of the Sarcosporidia. Z. Parasitenkd. 48 (1975) 73-82
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Heydorn, A. G. und S. Haralambidis: Zur Entwicklung von Sarcocystis capracanis Fischer 1979. Bed. Munch. tieriirztl. Wschr. 95 (1982) 265-271
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Mehlhorn, H. and A. G. Heydorn: Electron microscopical study on gamogony of Sarcocystis suihominis in human tissue cultures. Z. Parasitenkd. 58 (1979) 97-113 Mehlhorn, H., A. G. Heydorn,]. K. Frenkel, and E. Gobel: Announcement of the establishment of neohepantotypes for some important Sarcocystis species. Z. Parasitenkd. 71 (1985) 689-692
Mehlhorn, H, D. Diiwel und W. Raether: Diagnose und Therapie der Parasiten von Haus-, Nutz- und Heimtieren. G. Fischer, Stuttgart (1986)
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Mehlhorn, H. und E. Scholtyseck: Elektronenmikroskopische Untesuchungen an Cystenstadien von Sarcocystis tenella aus der Oesophagusmuskulatur des Schafes. Z. Parasitenkd. 41 (1973) 291-310 Mehlhorn, H. und E. Scholtyseck: Licht- und elektronenmikroskopische Untersuchungen an Entwicklungsstadien von Sarcocystis tenelfa aus der Darmwand der Hauskatze. I. Die Oocysten und Sporocysten. Z. Parasitenkd. 43 (1974) 251-270
Senaud, ].: Contribution it l'etude des sarcosporidies et des toxoplasmes. Protistologica 3 (1967) 167-232 Dr. Alfred Otto Heydorn, Inst. f. Parasitologie und Tropenveteriniirmedizin, FU Berlin, K6nigsweg 65, D-1000 Berlin 37