Oocyst Wall Formation Research Articles

ULTRASTRUCTURAL STUDIES ON THE ENDOGENOUS DEVELOPMENT OF EIMERIA BRUNETTI

The ultrastructural changes occurring during wall formation in the oocysts of E. brunetti were studied in tissue from the small intestine of young domestic fowls. The process of oocyst wall formation was initiated by a separation of the limiting membranes of the cytoplasmic mass of the macrogamete, to form a loose veil, consisting of 2-3 membranes, around the organism. Concomitant with the appearance of the veil, changes were observed in the Wall Forming Bodies of Type I (WFB I) and Type II (WFB II) which were present in the cytoplasm of the macrogamete. The outer layer of the oocyst wall seemed to be formed by a combination of membranes released from the surface of the organism and the contents of the WFB I. A 700 nm thick layer was thereby formed and, at this stage, the WFB I had disappeared from the cytoplasmic mass. The outer layer further condensed into a structure 250-350 nm in thickness in which 3 zones of different densities could be distinguished. The formation of the inner layer of the oocyst wall appeared to be associated with material from the WFB II and these disappeared from the cytoplasmic mass as the inner layer became evident. This inner layer was homogeneous in appearance and 80 nm thick. Comparison of the ultrastructure of the walls of oocysts obtained from chicken faeces with that of oocysts observed within the intestine showed only minor differences. The loose veil was not present in oocysts isolated from faeces and some changes were also noted in the outer zone of the outer layer of the oocyst wall. These differences could result from the mechanical and chemical treatments used for processing the faeces.

The ultrastructural development of the macrogamete and formation of the oocyst wall of Toxoplasma gondii.

The macrogametes of Toxoplasma gondii develop within the epithelial cells of the cat ileum. As they develop the nucleus enlarges and dense patches of chromatin which were present in the nucleoplasm, disappear. Polysaccharide granules and lipid globules appear in the cytoplasm and increase in number during development. The wall-forming bodies of Type I (WFB I) appear before the wall-forming bodies of Type II (WFB II); WFB I are smaller, more osmiophilic and more numerous than the WFB II. The WFB I appear to form from vesicles produced by the smooth endoplasmic reticulum, and the WFB II form within the lacunae of the rough endoplasmic reticulum. Double membraned vacuoles appear to form from the nuclear membranes but the function of these is unknown. Throughout development the macrogamete retains a normal pellicle which possesses numerous micropores. The first evidence of oocyst wall formation is the appearance of participate matter in the parasitophorous vacuole which precipitates to form Layer 1 of the oocyst wall. Layers 2 and 3 are unit membranes which form between Layer 1 and the pellicle. During this development the organism has an organelle complement similar to that of the macrogamete. Layers 4 and 5 form between Layer 3 and the pellicle. Layer 4 is osmiophilic and its formation is accompanied by the disappearance of WFB I. Layer 5 is less osmiophilic than Layer 4 and its formation is accompanied by the disappearance of WFB II. The two innermost layers (Layers 4 & 5) in the oocyst wall of Toxoplasma are similar to those found in Isospora spp. and Eimeria spp. Toxoplasma seems to be unusual in that, firstly, it possesses an additional 3 layers and, secondly, all 5 layers are formed outside the pellicle of the original macrogamete.

Development of second-generation schizonts, gamonts and oocysts of Eimeria bovis in bovine kidney cells.

First-generation merozoites of Eimeria bovis, obtained from calves which had been inoculated 14, 14 1/2 and 15 days earlier, were inoculated into monolayers of Madin-Darby bovine kidney (MDBK) and primary embryonic bovine kidney (BEK-1), as well as kidney cell aggregates (BEK-2). Cultures were examined at intervals of 1–12 hr for 168 hr with phase-contrast microscopy and then fixed, stained and re-examined with bright-field microscopy. Fourteen-day merozoites entered all cell types, whereas 14 1/2- and 15-day merozoites did not enter any. Development beyond the first-generation merozoite stage occurred in BEK-1 and BEK-2, but not MDBK. Intermediate and mature second-generation schizonts with 24–36 merozoites were seen at 48–108 hr. Merozoites were 4.5 by 1.4 μm, with a posteriorly located nucleus. Young, intermediate, and mature microgamonts were seen at 72–96 hr, 72–120 hr, and 96–120 hr, respectively. Young, intermediate, and mature macrogamonts were seen at 48–96 hr, 72–120 hr, and 96–120 hr, respectively. Mature microgamonts and macrogamonts were 14.7 μm in diameter and 19.5 by 15 μm, respectively. At 120–168 hr, zygotes in various stages of oocyst wall formation and oocysts (24.4 by 15 μm) were seen. A crescent body was present in the parasitophorous vacuole of some schizonts and gamonts.

Electron microscope studies of macrogametes and fertilization in Eimeria bovis.

Mature macrogametes of Eimeria bovis in the mucosa of the colon of calves killed 17 days after inoculation had a characteristic fine structure. Numerous osmiophilic wall-forming bodies of type I were located peripherally in the macrogamete, whereas the wall-forming bodies of type II were relatively large and located more centrally. Numerous polysaccharide bodies and lipid inclusions were also present. The parasitophorous vacuole, when present, was narrow and had electron-pale contents. The host cell cytoplasm was vacuolated; in some specimens the host cell appeared to have disintegrated. A microgamete was observed within a vacuole in the cytoplasm of a macrogamete from a calf killed 19 days after inoculation. This microgamete had two flagella and a homogeneous osmiophilic nucleus. The wall-forming bodies of type I and type II were altered in this macrogamete as compared with those of mature macrogametes, indicating that oocyst wall formation had begun. Many microgametes were observed in the cytoplasm of the host cell as well as in the parasitophorous vacuole of the macrogamete.