Figure 215

(A) Early stage of oocyst wall formation showing the outer veil (V) and partial formation of the outer layer of the oocyst wall (arrows). Note this is associated with the loss of the VFB and the WFB1 from the macrogamete cytoplasm, while the WFB2 (W2) remain. L, lipid droplet; PG, polysaccharide granule; N, nucleus. Bar = 1 |im.

(B) Newly released oocyst showing the outer veil (V) and fully formed oocyst wall (OW) enclosing a cytoplasmic mass containing polysaccharide granules (PG) and lipid droplets (L). Bar = 1 | m.

(C) SEM showing a number of microgametes (Mi) apparently attached to a macrogamete/ oocyst (Ma) with two adjacent merozoites (Me). Bar = 1 | m.

(D) Detail of the oocyst wall consisting of the outer veil (V) plus the thin, electron-dense outer layer (O) and the thicker inner layer (I), which is separated from the plasmalemma (P) of the cytoplasmic mass. Bar = 100 nm.

and coalesce to form the electron-lucent inner layer of the oocyst wall (layer 5; Figures 2.15B, 2.15D) (Ferguson et al., 1975). The cytoplasmic mass loses the WFBs during oocyst wall formation and is characterized by a central electron-lucent nucleus and cytoplasm packed with polysaccharide granules and lipid droplets (Figure 2.15B).

This process is identical to that described for the closely related genus Eimeria (Ferguson et al., 2003). For correct formation of the oocyst wall there is a requirement for tight control and sequential secretion of the veil-forming bodies and the wall-forming bodies 1 and 2. From the available data for Coccidia it would be most accurate to consider the outer veil as part of the early development, as it is lost by the time oocysts are released with the feces. The oocyst proper can be considered as a double-layered structure (reviewed by Belli et al., 2006). The outer electron-dense layer is thinner in the T. gondii oocyst (Figure 2.15D) than in those of Eimeria spp. (Belli et al., 2006). The formation and polymerization of the inner layer has a dramatic effect on the ability to process the oocyst for ultrastructural examination. To date, no technique has been developed that will allow the oocysts of T. gondii or any other coccidian oocyst to be examined by electron microscopy. Over the past 30 years, numerous attempts, using many electron microscopic fixatives and embedding protocols, have resulted in failure. The two layers provide different structural and chemical protection. The outer layer contains mostly proteins and carbohydrate, and appears to provide structural strength. In contrast, the inner layer has a high lipid content and appears to provide the protection from chemical insult by its impervious nature (even to electron-microscopy reagents). Work on the properties of the oocyst is continuing in the closely related genus Eimeria (Belli et al., 2006).

2.3.5 Fertilization

It would appear logical that if sexual development takes place, there will be fusion between a microgamete and a macrogamete to form a fertilized zygote. However, this process has never been visualized. It could be expected that the mature microgametes and macrogametes are released from the host cells and fertilization takes place in the lumen. Indeed, macrogametes/oocysts with attached microgametes have been observed on rare occasions (Figure 2.15C) (Ferguson, 2002). However, as has been described above, oocyst wall formation is initiated prior to release of the macrogamete from the host cell. An additional anomaly in T. gondii is the formation of very few microgametocytes, so there are relatively few microgametes in relation to the number of macrogametes. It is a universal feature of plants and animals that there is a vast excess of male gamete formation because of the importance of ensuring maximum fertilization of the female gametes. That fertilization can occur has been proven from the identification of cross-fertilized parasites (Pfefferkorn and Pfefferkorn, 1980). However, T. gondii, unlike most other metazoans, is normally haploid, and whether this will affect the necessity for fertilization is open to question (Ferguson, 2002).

2.3.6 Oocyst and extracellular sporulation

The oocyst is the only stage of T. gondii that is capable of undergoing extra-cellular development - all other development processes can only occur within viable nucleated host cells. The oocysts are excreted in an unsporulated form with a single undifferentiated cytoplasmic mass - the primary sporoblast (Figure 2.16A). In the external environment asexual development (sporulation) occurs, which finally results in the formation of two sporocysts, each of which contains four sporo-zoites. Initial attempts to study this process were unsuccessful because of the inability to process the oocyst for ultrastructural examination. It was only possible to overcome this problem by developing a technique that involved freezing and cryosectioning of the oocysts prior to processing for electron microscopy (Birch-Andersen et al., 1976). The aim was to fracture the oocyst wall without destroying the cytoplasmic mass within. This technique is inefficient, with destruction of a large proportion of oocysts; however, a few oocysts remain intact and these are used to examine the ultrastructural changes associated with sporulation. Due to these difficulties, studies have been limited to a series of papers on the sporulation of E. brunetti (Ferguson et al., 1978a, 1978b) as a model for the genus Eimeria and T. gondii

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