Fine-Structural Aspects of Macrogametogenesis in Eimeria magna*†

SYNOPSIS. Macrogamonts in tissues from rabbits killed 5 1/2 days after inoculation with Eimeria magna oocysts were studied with the electron microscope. In young macrogamonts, parts of cytoplasm, sometimes including micronemes, were pinched off into the parasitophorous vacuole. In all stages of development, small segments of the inner membrane complex were present beneath the limiting membrane. Micropores also were seen in all stages, and some apparently functional ones were present in mature macrogametes. Wall-forming bodies of Type I and Type II were observed in relatively early stages. The former were less numerous than the latter, which had a more compact appearance than in other species. Usually, several Golgi complexes were present and several Golgi adjuncts occurred in the vicinity of the nucleus in all stages of development. Microgametes were observed in the cytoplasm of host cells harboring immature macrogametes.     

The ultrastructure and distribution of micropores in the various developmental forms of Eimeria brunetti

The structure and distribution of micropores in the various developmental stages of Eimeria brunetti was examined. Micropores were observed in all the endogenous forms with the exception of the microgamete. Oocysts from chicken faeces were also examined at various stages of sporulation and micropores were demonstrated in zygotes, sporoblasts, sporozoites, and the residual cytoplasmic masses. The number of micropores per organism appeared to be correlated with the surface area of the organisms irrespective of whether these were endogenous or sporulating forms. The increase in the number of micropores did not appear to be related to micropore activity because semmingly active micropores were observed only in the trophozoites, in the early multinucleate forms (early shizonts and microgamonts), and in the early macrogamonts. All these forms, however, possessed relatively few micropores. No active micropores were ever observed within the sporulating oocysts.     

Observations on the Endogenous Stages of Eimeria confusa Joseph, 1969 from the Grey Squirrel Sciurus carolinensis*

SYNOPSIS. Stages in the endogenous cycle of Eimeria confusa from the grey squirrel, Sciurus carolinensis, are described from mixed infections with another species, Eimeria lancasterensis. All corresponding stages were markedly different in the 2 species. In E. confusa infections, the parasites were located below the host cell nuclei of the epithelial cells of the villi of the jejunum and ileum. Mature schizonts were ellipsoidal, averaged 20.9 × 18.6 μm and had 18–30 merozoites. The mature microgamonts measured 34.3 × 24.7 μm and had hundreds of microgametes. Mature macrogametes were ovoid, averaged 31.3 × 25.6 μm, and contained 2 kinds of plastic granules.     

The fine structure of the endogenous stages of Eimeria labbeana: 2. Mature macrogamonts and young oocysts.

The fine structure of the mature macrogamonts and intracellular oocysts of Eimeria labbeana from the ileal mucosa of experimentally infected Pigeons (Columbia livia) was investigated and described. The macrogamont reached a maximum size of 12.0 x 9.5 mum (average equals 10.8 x 8.8 mum), and was located within a narrow parasitophorus vacuole. Most of the macrogamonts were limited by two membranes. Intravacuolar tubules, 1.2 mum long and 58 nm in diameter, established direct connections between the parasite and the host cell. Each tabule was composed of 9 subunits arranged around the central lumen. Cytoplasmic canaliculi were composed of bundles of microtubule-like structures (8-10 nm wide). Type 1 wall-forming bodies reached a maximum size of 1.8 x 1.5 mum, and many had centric or eccentric electron transparent portions within them. They were frequently seen lodged within peripherally-located mitochondria. Type 2 wall-forming bodies averaged 1.5 mum in diameter. The role of the two types of wall-forming bodies in forming the outer and inner layers of the wall of the oocyst was similar to that in other species of Eimeria. The oocyst wall was 0.2 mum thick and composed of a limiting membrane (20 nm thick), an outer layer (75 nm thick), and an inner layer (100 nm thick).     

The life cycle of Eimeria falciformis var. pragensis (Sporozoa: Coccidia) in the mouse, Mus musculus

The life cycle of Eimeria falciformis var. pragensis, established from a single oocyst, is described in experimentally infected mice (Mus musculus). The coccidium had a prepatent period of 7 days and a patent period of 10--16 days. Oocysts were spherical to ellipsoidal in shape and measured 21.2 x 18.3 micron. Sporulation time was 3 to 3.5 days. Sporocysts measured 12.2 x 7.2 micron and contained a circular to avoid granular sporocyst residuum measuring 5.5 X 5.0 micron. One, 2 or 3 circular to rectangular polar granules were observed within each sporulated oocyst. The endogenous stages developed primarily in the cecum and colon and only occasionally in the lower ileum. Four generations of schizonts were found. Mature 1st-generation schizonts, first observed 48 hr postinfection (PI), measured 17.8 x 12.3 micron and had 12 merozoites that measured 13.3 x 2.0 micron. Mature 2nd-generation schizonts appeared 78 hr PI. They measured 10.2 x 9.3 micron and had 8 merozoites measuring 5.0 x 1.6 micron. Mature 3rd-generation schizonts appeared first at 114 hr PI and measured 17.5 x 10.2 micron and had 10 merozoites that measured 12.4 x 1.8 micron. Mature 4th-generation schizonts appeared first at 144 hr PI. They measured 18.2 x 15.3 micron and had 18 merozoites. The merozoites of the 4th-generation schizont were 4.5 x 1.2 micron. Mature macrogamonts and microgamonts developed simultaneously appearing at 156 hr PI. Macrogamonts measured 16 x 14.5 micron and microgamonts were 18.2 x 15.3 micron. In experimentally infected rats (Rattus norvegicus), development of E. falciformis var. pragensis progressed only as far as mature 1st-generation schizonts.     

Fine Structure of the Endogenous Stages of Eimeria labbeana. I. The First Generation Merozoites

SYNOPSIS. The fine structure of the 1st generation merozoites of Eimeria labbeana from the ileal mucosa of artificially infected pigeons ( Columba livia ) was investigated and described. The 1st generation merozoites which appeared between 36-48 hr after infection averaged 4.4 × 2.1 μm in size. The 3-membraned pellicle was irregular in texture and harbored a single micropore, and many micropore-like invaginations. Closely apposed to the inner pellicular membrane were seen 22 microtubules, each 22–25 nm in diameter. An apical vesicle, 50 nm in diameter, seen at the anterior extremity, was connected with the common duct of the micronemes. The conoid consisted of 9 spiral elements, each 30 × 25 nm. The paired organelle (rhoptries) varied in length (1.4–2.2 μm), and the ductules (23 nm diameter) were composed of 2 inner tubules, each 6 nm in diameter. A unit membrane enveloped the partially alveolar and differentially osmiophilic interior of the bulbous regions of the rhoptries. The "rod-like structure"was found to be tubular and represented the common duct of the micronemes.     

In vivo action of the anticoccidial diclazuril (Clinacox) on the developmental stages of Eimeria tenella: an ultrastructural evaluation

A single 5-mg/kg oral dose of diclazuril affected both the asexual and sexual development of Eimeria tenella in experimentally inoculated chickens. In second-generation schizonts, early growth and nuclear divisions progressed normally, but a marked inhibition of merozoite formation was observed. Exogenesis of merozoites was largely prevented, whereas production of micronemes, amylopectin granules, and dense bodies and the formation of rhoptries, conoid, and pellicle continued. All these subcellular organelles accumulated, together with differentiated nuclei, within the main cytoplasmic mass. In the end, complete necrosis of the schizonts occurred. In macrogamonts, dilation of the rough endoplasmic reticulum around type II wall-forming bodies, fusion of type II wall-forming body contents, disturbance of the normal parallel arrangement of rough endoplasmic reticulum, and disruption of row formation of amylopectin granules became evident. In the microgamonts, normal evagination of microgametes was prevented; the flagellar complex formed within the main cytoplasmic mass and the differentiated nuclei remained present within the parasite body. The macro- and microgamonts also ended up in a stage of complete necrosis. These data indicate that diclazuril treatment primarily affects the normal differentiation of the respective endogenous stages during parasite development. This leads to complete degeneration of schizonts and gamonts indicating the lethal effect of this new anticoccidial compound.     

Comparative In Vitro Development of Precocious and Normal Strains of Eimeria tenella (Coccidia)

SYNOPSIS. Eimeria tenella strain Wis-F is known to develop in chickens with a significantly shortened prepatent period and its pathogenicity is virtually completely attenuated. In vitro development of this strain paralleled development of the control (Wisconsin) strain through the first asexual generation. Instead of entering 2nd generation schizogony, however, most of the Wis-F merozoites developed into microgamonts or macrogamonts. Wall-forming bodies were prominent in developing macrogametes at 80–88 hr and began coalescing into the oocyst wall by 88 hr. Microgamete development paralleled that of macrogametes, with the appearance of multinucleate, immature forms at 72–80 hr and with recognizable, spermlike microgametes being prominent at 88–96 hr. Pathogenicity attenuation and reduction of the length of the prepatent period clearly resulted from omission of a portion of the life cycle (2nd generation schizogony).     

Life Cycle of Eimeria ferrisi Levine & Ivens, 1965 in the Mouse, Mus musculus

SYNOPSIS. The life cycle of Eimeria ferrisi is described from experimentally infected Mus musculus. The prepatent period was 3 days and the patent period was 3–4 days. The endogenous stages were found only in the cecum and colon. Three generations of schizonts were found. Mature 1st-generation schizonts first seen 24 hr postinoculation (PI) measured 10.9 (7–14) × 10.2 (6–13) μm and had 9.6 (7–14) merozoites. Some 2nd-generation schizonts had uninucleate merozoites and others had multinucleate merozoites. The former were first seen in small numbers 36 hr PI and were most abundant 48 hr PI. They measured 9.6 (5–13) × 7.9 (6–12) μm and had 18 (6–25) merozoites. Schizonts with multinucleate merozoites were seen 72 hr PI. Mature 3rd-generation schizonts were seen 72 hr PI. They measured 14.0 (12–18) × 11-0 (9–13) μm and had 12.5 (5–16) merozoites. Macrogamonts were first seen in 72 hr sections. Each young macrogamont had a large nucleus with a prominent nucleolus. Only one type of cytoplasmic granule appeared to be involved in the formation of the oocyst wall. Mature macrogamonts were 11.0 (5–14) × 10.0 (6–13) μm. Crescent-shaped bodies were observed in the parasitophorous vacuole of trophozoites and young macrogamonts. Early microgamonts were first recognized at 96 hr by the presence of darkly stained and irregularly shaped nuclei. Usually, mature microgametes were arranged in long, narrow whorls at the periphery of the microgamont or in whorls at the surface of 2–5 compartments.     

Further studies on the development of gamonts and oocysts of Eimeria magna in cultured cells

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.     

Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites

The ultrastructural characterisitics of four types of Toxoplasma gondii schizonts (types B, C, D and E) and their merozoites, microgamonts and macrogamonts were compared in cats killed at days 1, 2, 4 and 6 after feeding tissues cysts from the brains of mice. Schizonts, merozoites and gamonts contained most of the ultrastructural features characteristic of the phylum Apicomplexa. All four types of schizonts developed within enterocytes or intraepithelial lymphocytes. Occasionally, type B and C schizonts developed within enterocytes that were displaced beneath the epithelium into the lamina propria. Type D and E schizonts and gamonts developed exclusively in the epithelium. Tachyzoites occurred exclusively within the lamina propria. Type B schizonts formed merozoites by endodyogeny, whereas types C to E developed by endopolygeny. The parasitophorous vacuoles surrounding type B and C schizonts consisted of a single membrane, whereas those surrounding types D and E schizonts were comprised of two to four electron-dense membranes. The parasitophorous vacuole of type B schizonts had an extensive tubulovesicular membrane network (TMN); the TMN was reduced or absent in type C schizonts and completely absent in types D and E schizonts and gamonts. Type B merozoites were ultrastructurally similar to tachyzoites, except that they were slightly larger. Type C merozoites exhibited a positive periodic acid-Schiff reaction by light microscopy and ultrastructurally contained amylopectin granules. Rhoptries were labyrinthine in type B merozoites but were electron-dense in types C-E. The development of microgamonts, macrogamont and oocysts is also described.     

The sexual stages of Eimeria wyomingensis Huizinga and Winger, 1942, in experimentally infected calves

Seven of 12 calves given 10(6) Eimeria wyomingensis sporulated oocysts had sexual stages of the parasite when examined at necropsy. Clinical signs of coccidiosis were not observed in any calf. Sexual stages were located in host cells in the lamina propria of the villi in the terminal small intestine. Infected host cells underwent nuclear and cytoplasmic hypertrophy. Immature microgamonts usually had folded cytoplasm and an overall spherical to elongate shape. Mean length and width +/- SEM of immature microgamonts were 43.3 +/- 1.6 by 29.0 +/- 1.1 micron. Mature microgamonts contained hundreds of microgametes, lacked visible cytoplasmic folds, and measured 52.8 +/- 4.7 by 43.0 +/- 4.2 micron. Macrogamonts were spherical to ovoid and had a large nucleus and prominent nucleolus. Immature macrogamonts without visible wall-forming bodies measured 16.0 +/- 0.5 by 13.3 +/- 0.2 micron. Mature macrogamonts had 3-8-micron eosinophilic wall-forming bodies and measured 24.6 +/- 0.7 by 19.6 +/- 0.8 micron. Oocysts were ovoid and had a 2-3-micron-thick eosinophilic oocyst wall. A micropyle was present in appropriately sectioned oocysts. Oocysts measured 27.7 +/- 1.7 by 19.3 +/- 0.8 micron. The sexual stages of E. wyomingensis are compared to those described previously for species of Eimeria infecting the bovine small and large intestines.     

Fine Structure of Microgametocytes and Macrogametes of Eimeria nieschulzi

SYNOPSIS. An electron microscope study of microgametocytes and macrogametes of Eimeria nieschulzi Dieben, 1924 revealed that they lie within vacuoles bounded by a host unit membrane. The vacuole surrounding the microgametocyte contains granular material. The vacuole around the macrogamete is narrower and contains vesicles and membranes. Micropores were seen on the surface of the plasma membrane of microgametocytes and macrogametes. Microtubules were seen in macrogametes. Young microgametocytes and macrogametes have a similar cytoplasmic matrix, mitochondria and nuclei. Glycogen granules apparently develop around vacuoles in both microgametocytes and macrogametes. Glycogen granules were also seen along the margins of parallel bundles of fibers in microgametocytes. As nuclei of the microgametocyte divide, they move to the periphery of the parasite. Three basal bodies, each with 9 fibers in triplet form, develop in association with each nucleus. Microgametes have 2 free flagella and a central short, attached flagellum. Basal granules lie along the outer fibers of the central flagellum. Each microgamete has an elongate mitochondrion in close contact with the nucleus. In macrogametes wall-forming bodies develop in lacunae in the cytoplasm. Smaller dark bodies with areas of low density were also seen. Wall-forming bodies and dark bodies move to the periphery of mature macrogametes.     

Endogenous stages of Eimeria tuskegeensis (Protozoa: Eimeriidae) in the cotton rat, Sigmodon hispidus

Endogenous stages of Eimeria tuskegeensis were studied in experimentally infected cotton rats, Sigmodon hispidus. Almost all parasites were located on the basilar side of the nucleus in epithelial cells on the sides and tips of villi of the small intestine. The endogenous cycle consisted of three generations of schizogony followed by gametogony. First-, second-, third-generation schizonts could be distinguished by time of appearance, size and shape of the schizont, and number, size, shape, and arrangement of merozoites. Immature gametogonous stages appeared to 84 hr postinoculation (PI) and developed into mature microgametocytes and macrogametes by 96 hr PI. Microgametocytes had a mono-centric type of development. Intermediate macrogametes had small, basophilic wall-forming bodies and mature macrogametes had large, eosinophilic wall-forming bodies. It was not possible to determine whether these were two distinct types of wall-forming bodies or whether they were different stages of a single type. Two nuclei were seen in the host's epithelial cells parasitized by schizonts, microgematocytes, macrogametes, and oocysts. This binucleate condition was apparently parasite-induced.     

The ultrastructure of gametogenesis of cryptosporidium baileyi (eimeriorina; cryptosporidiidae) in the respiratory tract of broiler chickens (Gallus domesticus).

The ultrastructural features of sexual development of Cryptosporidium baileyi in the respiratory tract of experimentally infected broiler chickens were studied using transmission electron microscopy. Sexual stages of C. baileyi were seen attached to the tracheal epithelium and free in the tracheal lumen. These stages included intracellular type III merozoite-like stages, microgamonts, microgametes, macrogamonts, thin-walled oocysts, and thick-walled oocysts. These stages were developmentally similar to those observed for other Cryptosporidium species. All of the above stages were observed during each study day. Thin-walled oocysts, microgamonts, and microgametes were seen less frequently than other sexual stages. Microgamonts, macrogamonts, and oocysts attached to the epithelium were all contained in a host cell membrane or within a parasitophorous vacuole. Thin-walled oocysts of C. baileyi were observed for the first time on an ultrastructural level in the respiratory tract of chickens.     

Eimeria apsheronica in the goat: endogenous development and host cellular response

Most first generation schizonts of Eimeria apsheronica developed in the jejunum; others were distributed throughout the small intestine and occasionally in the caecum. Some were also found in the mesenteric lymph nodes, which were oedematous and haemorrhagic. In the intestine, haemorrhage and congestion were seen before parasites were detected, and continued throughout all later stages. Schizonts occurred in the lamina propria and occasionally in the submucosa, where they sometimes caused a cellular inflammatory response. Schizonts were first seen at 8 days post-infection (DPI); they had poorly defined nuclei and were enclosed in a capsule-like wall. At 16 DPI, many had matured, had a mean size of 125 x 82 microns, and were filled with numerous spindle-shaped merozoites, which were in ranks and loops. At 18 and 20 DPI, when small white lesions (1-3 mm in diameter) were observed in the jejunum and elsewhere in the small intestine, a second generation of schizonts, macrogametes, microgametocytes and maturing oocysts were seen, in the epithelial cells of the small intestine and caecum. Their mean sizes, respectively, were: 26.2 x 18.9, 24.7 x 18.5, 30.2 x 21.7 and 26.6 x 19.3 microns. Macrogametes contained basophilic central and eosinophilic peripheral granules. The sexual stages were associated with a generalized cellular inflammatory response.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.     

Development of Eimeria meleagrimitis Tyzzer from sporozoites and merozoites in turkey kidney cell cultures

Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P less than 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P less than 0.05) than their in vivo counter parts.     

Fine Structures Associated with Nutrition of the Intracellular Parasite Eimeria auburnensis*

SYNOPSIS. In the nearly mature macrogametes of Eimeria auburnensis, the cell membrane is a unit membrane, with underlying and overlying osmiophilic layers usually present. Cup-shaped micropores were occasionally seen. Smaller, V-shaped invaginations were also found in considerable numbers at the surface. At the deepest point, these invaginations were bounded only by a unit membrane. Immediately adjacent to this point, vesicles with homogenous electron-pale contents bounded by a similar unit membrane, were frequently seen. Pinocytosis evidently occurs at the site of these invaginations. Numerous folds of the host cell membrane bordering the vacuole in which the parasite lay extended about 0.1–0.7 μ into the vacuole. These “intravacuolar folds” varied in depth and number in different specimens. In some, the majority of folds had apparently become disconnected from the host cell membrane. A highly developed smooth endoplasmic reticulum occurred in the adjacent host cell cytoplasm. The intravacuolar folds may assist in transfer of nutrients, including membrane material, from the host cell to the parasite. The evidence indicates that in this species of Eimeria nutrients are taken into the parasite primarily as fluids by pinocytosis and possibly other processes.     

Fine-Structural Aspects of Microgametogenesis of Eimeria magna in Rabbits and in Kidney Cell Cultures*

SYNOPSIS. The fine-structural aspects of development of microgamonts of Eimeria magna were studied in kidney cell cultures and in experimentally infected rabbits. Spheroidal masses of gamont-like cytoplasm containing ribosomes, polyribosomes, and amylopectin granules were found within the parasitophorous vacuole; these bodies were apparently pinched off the surface of the gamont. Nucleoli were present in the early stages of nuclear division but disappeared as development proceeded. Spindles were eccentric and the nuclear membrane always remained intact in dividing nuclei. Nuclei eventually became elongate in shape, compact, and electron dense at the end oriented toward the periphery and lucent centrally. Usually, only the dense portion was incorporated into the gamete as the gamete developed by protruding from the gamont surface. Fullyformed microgametes were biflagellate and contained a nucleus, a mitochondrion, and 8-10 microtubules. Multiple-membrane complexes, which apparently originated from the nuclear envelope or endoplasmic reticulum, were found within the gamont residual body. Occasionally, 2 or 3 micro- and/or macrogamonts were seen within the same cell. In some cells one or 2 micro- or macrogamonts as well as several merozoites were present in the same parasitophorous vacuole.