Figure 211

(A) Late schizont showing the daughters filling the mother cell cytoplasm and the partial invagination of the plasmalemma around the daughters (arrows). N, nucleus; R, rhoptry; MN, microneme. Bar = 1 |im.

(B) Mature schizont with fully formed merozoites containing the characteristic apical organelles. N, nucleus; C, conoid; R, rhoptry; DG, dense granule. Bar = 1 |im.

(C) Scanning electron micrograph of a fracture through an intestinal villus from an infected cat. A number of small trophozoites (Tr) and two mature schizonts with crecentic shaped mero-zoites (arrows) can be seen within the epithelial cells. Bar = 2 | m.

the nucleus. The merozoites often remain attached to a small amount of residual cytoplasm at the posterior end. These banana-shaped daughters can be seen forming fan-like structures (Figures 2.11B, 2.11C). The merozoites are released from the host cell into the lumen, where they can reinvade enterocytes. However, this process has not been observed by electron microscopy.

Unlike many Eimeria species, there does not appear to be the distinct sequential generations of schizogony, which differ from each other in their size and number of daughters formed. However, in studies of the early stages of infection (1-3 days), additional asexual processes have been described (Speer and Dubey, 2005). It has been reported that certain developing parasites have a similar host-parasite relationship and undergo endodyo-geny and repeated endodyogeny (type B schizonts), while others appeared intermediate (type C schizonts). The type B schizonts have a similar relationship to that described for parasites invading the small intestine of the intermediate host (Dubey, 1997; Speer and Dubey, 1998). These stages appear to be rare, and could represent examples where the initial invading bradyzoite failed to undergo conversion to coccidian development but underwent conversion to tachyzoite development, as seen in the intermediate host. It is known that the cat can act as an intermediate host as well as the definitive host.

2.3.3 Sexual development

After an unknown number of asexual cycles, certain merozoites on entering a new enterocyte can develop into either male (microgametocyte) or female (macrogametocyte) gametocytes. In microgametogony this results in the formation of multiple (15-30) male gametes (microgametes) and in macrogametogony the formation of a single female gamete (macrogamete). The trigger for the conversion from asexual to sexual development is unknown; nor is it known what is responsible for deciding whether an invading merozoite develops into either a microgametocyte or macrogametocyte.

The initiation of gametocyte formation appears to be less controlled in T. gondii than in other species of Coccidia. In the majority of Eimeria spp. there is a fixed number of sexual cycles followed by the vast majority of merozoites simultaneously developing into sexual stages. In T. gondii there does not appear to be a distinct conversion, with a mixture of both asexual and sexual stages observed throughout enteric development. There is no ultrastructural feature that could identify the merozoites that will develop into sexual stages, nor are there any differences in the host-parasite relationship or parasitophorous vacuole.

On entering the host cell the merozoite becomes more spherical and loses the majority of its apical organelles, such as the rhoptries and dense granules, although the conoid and a few micronemes remain. This stage (trophozoite) begins to grow and there appears to be an increase in the size of the mitochondrion/mitochondria, which are located round the periphery. However, at this stage it is not possible to differentiate between organisms that will develop into micro- and macro-gametocytes. Microgametogony and the microgamete

There are relatively few descriptions of microga-metogony (Colley and Zaman, 1970; Pelster and Piekarski, 1971; Ferguson et al., 1974; Dubey et al., 1998). Initially is impossible to differentiate between the proliferative phase of endopolygeny and microgametogony, with both processes involving continued growth and repeated nuclear divisions. It has been reported that the earliest stage allowing differentiation between schizogony and microgametogony is based on differences in the distribution of the nuclear chromatin (Ferguson et al., 1974). During schizogony the electron-dense heterochromatin remains dispersed throughout the nuclei (Figure 2.9E), whereas in the later stages of microgametogony the heterochro-matin condenses into electron-dense masses at the periphery of the nucleus (Figure 2.12A). In microga-metogony, the nuclei move to the periphery of the cell with two centrioles and a dense plaque (perforatorium) located between the nuclei and the plasmalemma (Figure 2.12A). The centrioles become

FIGURE 2.12 Electron micrographs illustrating various stages in the process of microgametogony.

(A) Mid-stage microgametocyte showing the peripherally located nuclei containing areas of condensed chromatin (arrows). The centriole (Ce) and the plate-like perforatorium (P) can be seen located between the nuclei (N) and the plasmalemma. Bar = 1 |im.

(B) Detail showing the protrusion the flagella (F) plus the dense portion of the nucleus and a mitochondrion (Mi) into the PV. Bar = 0.5 |im.

(C) Late stage showing a number of microgametes forming in the PV while still attached to the mother cell (arrows). N, nucleus; F, flagellum. Bar = 1 | m.

(D) Detail from Figure 2.12C showing elongating nucleus (N) and mitochondrion (Mi) of the microgamete still connected to the mother cell (arrows). F, flagellum. Bar = 0.5 | m.

the basal bodies for the developing flagella, which begin to grow by protruding into the parasitophorous vacuole (Figure 2.12B). Interestingly, although the centrioles differ from metazoan centrioles, the flagella have the typical nine peripheral doublet tubules with the two central microtubules. As this flagellar growth occurs, the chromatin condensation continues at the side of the nucleus closest to the centrioles, with the other part of the nucleus having a more electron-lucent appearance. In addition, a mitochondrion is located adjacent to each nucleus (Figure 2.12B). There is no significant development in the apicoplast during this process (Ferguson et al., 2005). Microgamete development continues with flagellar growth and protrusion of a portion of cytoplasm containing the basal bodies, the electron-dense portion of the nucleus, and a mitochondrion into the lumen of the PV (Figures 2.12B, 2.12C, 2.12D). As this occurs there is division of the nucleus, with the electron-dense portion separating from the electron-lucent portion. The electron-dense portion enters the developing microgamete, and the lucent portion remains within the mother cell as a residual nucleus. The microgametocyte of T. gondii produces relatively few (15-30) microgametes (Figure 2.12C). The immature microgametes are still attached to the mother cell by a narrow cytoplasmic isthmus (Figures 2.12C, 2.12D).

Maturation continues with each microgamete becoming elongated in appearance and consisting of an electron-dense nucleus with a mitochondrion located between the nucleus and the basal bodies from which the two very long flagella run toward the posterior (Figures 2.13A, 2.13B). In addition there is an electron-dense plate termed the perforatorium in the apex and a number (four) of longitudinally running microtubules (Ferguson et al., 1974). Once fully formed the microgametes detach from the microgametocyte, leaving a large residual cytoplasmic body. Macrogametogony and the macrogamete

The development of the macrogametocyte has been described in a few studies (Colley and Zaman, 1970; Pelster and Piekarski, 1972; Ferguson et al., 1975). It is associated with growth of the trophozoite and the appearance of a large nucleus with

FIGURE 2.13 The structure of the mature microga-


(A) Longitudinal TEM section through a microga-mete showing the electron-dense nucleus (N) and the anterior mitochondrion (Mi) and the basal bodies of the two flagella (F). Bar = 1 |im.

(B) SEM of a microgamete illustrating the nucleus (N) and the two very long, posteriorly pointing flagella (F). Bar = 1 |im.

FIGURE 2.13 The structure of the mature microga-


(A) Longitudinal TEM section through a microga-mete showing the electron-dense nucleus (N) and the anterior mitochondrion (Mi) and the basal bodies of the two flagella (F). Bar = 1 |im.

(B) SEM of a microgamete illustrating the nucleus (N) and the two very long, posteriorly pointing flagella (F). Bar = 1 |im.

dispersed chromatin and a large nucleolus but no nuclear division. As the macrogametocyte grows there is a marked increase in the size of the peripherally located mitochondrion and the centrally located apicoplast (Figure 2.14A). In addition, a number of Golgi bodies are distributed throughout the cytoplasm. The first distinct organelle of macrogametogony is the appearance of flocculent material condensed within dilatations of the rER (Figures 2.14B, 2.14D). This material represents the initiation of formation of the wall-forming body type 2 (WFB2), such bodies being so called because of their role in the formation of the oocyst wall (see below). A Golgi body is often associated with the membrane of ER surrounding the wall-forming bodies type 2. As maturation continues, there is an increase in size and number of the wall-forming bodies type 2, and a number of electron-dense membrane-bound granules appear to form from vesicles produced by the Golgi bodies (Figure 2.14C). These are of various sizes, and are termed wall-forming bodies type 1 (WFB1) (Figure 2.14D). However, it has been

FIGURE 2.14 Various stages in the development of the macrogametocyte.

(A) Early macrogametocyte characterized by the central nucleus (N) with a large nucleolus (Nu). The cytoplasm contains peripheral profile of an enlarged mitochondrion (Mi) and an enlarged Golgi body (G). A few polysaccharide granules (PG) and lipid droplets were present in the cytoplasm. Bar = 1 | m.

(B) Mid-stage macrogametocyte showing increased numbers of polysaccharide granules (PG) and lipid droplets (L), the appearance of wall-forming bodies type 1 (W1) and type 2 (W2) in the cytoplasm, and an increase in size of the apicoplast, A. Bar = 1 | m.

(C) Mature macrogamete showing the centrally located nucleus with adjacent apicoplast, A. The cytoplasm contains numerous wall-forming bodies type 1 (W1) and a few type 2 (W2), plus numerous polysaccharide granules (PG) and lipid droplets (L). Bar = 2 |im.

(D) Detail of the cytoplasm of a mature macroga-mete showing the numerous dense granules representing the wall-forming bodies type 1 (W1). The wall-forming body type 2 (W2) is located within the rough endoplasmic reticu-lum. PG, polysaccharide granule; L, lipid droplet. Bar = 0.5 |im.

possible, using immuno-electron microscopy, to identify two populations of membrane-bound electron-dense granules (Ferguson et al., 2000). One population appears to be involved in the formation of the outer veil, and consists of what are termed the veil-forming bodies (VFBs). These were originally termed wall-forming bodies type 1a, but, with the identification of similar granules in the macrogametocyte of Eimeria maxima, it is proposed that VFBs may be a more appropriate term (Ferguson et al., 2003). The wall-forming bodies type 1 appear slightly larger than the veil-forming bodies. As the veil- and wall-forming bodies are being synthesized, there is also the synthesis of numerous polysaccharide granules and lipid droplets and an expansion of the apicoplast (Figure 2.14C). When fully developed, the macrogametocyte can be considered to be a mature macrogamete (Figure 2.14C). This is not a sharp division but one of convenience to differentiate the developing stage from the mature gamete.

2.3.4 Oocyst wall formation

The oocyst wall is a multi-layer structure which is extremely resistant to physical and chemical insults. As such, it is fundamental to the survival of the parasite. Without this wall, the parasite could not survive for extended periods in the external environment required for transmission between hosts resulting from fecal contamination. The oocyst wall is a complex structure consisting of distinct layers (Ferguson et al., 1975; Speer et al., 1998), and is synthesized while the macrogamete is still within the host cell. In reviewing these data with reference to later observations for both T. gondii and Eimeria spp., the wall can be divided into three zones. The first is a loose outer veil consisting of two or three membranes (termed layers 1-3; Ferguson et al., 1975), which is formed by the fusion of the veil-forming bodies with the macrogamete plasmalemma and release of their contents (Ferguson et al., 2000). This occurs during the maturation of the macrogamete. This is followed by the triggered secretion of the WFB1, which occurs simultaneously in the mature macrogamete to form the outer layer of the oocyst wall, (Figure 2.15A) (layer 4; Ferguson et al., 1975). This initially forms a thick layer that undergoes polymerization to form a 30-70-nm electron-dense layer. Finally, the contents of the WFB2 are released

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