Date of Award:

5-1-1973

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Zoology

Committee Chair(s)

Datus M. Hammond

Committee

Datus M. Hammond

Committee

Thomas L. Bahler

Committee

Paul B. Carter

Committee

Hugh P. Stanley

Committee

Merthyr L. Miner

Abstract

Development of Eimeria ninakohlyakimovae from intracellular sporozoites to mature first-generation merozoites was studied. Monolayer cell line cultures of ovine trachea, kidney, and thyroid cells were inoculated with sporozoites of E. ninakohlyakimovae. Cultures containing parasites were fixed at the appropriate number of days after inoculation and prepared for examination. Sporozoites, trophozoites, schizonts, and merozoites grown in cultured cells were examined with the electron microscope. Intracellular sporozoites had ultrastructural characteristics similar to those of extracellular sporozites, except that apparently functional micropores were observed. During transformation of sporozoites to trophozoites, the conoid, annuli, and subpellicular tubules, as well as some portions of the inner membrane complex of the pellicle, disappeared. Trophozoites and binucleate schizonts had a reduced number of micronemes and rhoptries. In schizonts with three nuclei, only a few remnants of the micronemes and rhoptries were seen and the inner membrane comples had almost completely disappeared. One such schizont had one or more centrioles associated with each nucleus; in one, an eccentric spindle with a dense, cone-shaped structure at each pole was observed. A moderately dense, granular crescent body, probably derived from host cell material, was seen within the parasitophorous vacuole in some specimens. Larger schizonts in which a number of nuclei were present had only a single outer limiting membrane. Micropores observed in schizonts were not as greatly expanded as some seen in sporozoites. The numbers of mitochondria and ribosomes appeared to increase as the cytoplasmic volume of schizonts increased. A paras itophorous vacuole surrounded all schizogonous stages, including immature and nearly mature schizonts. The cytoplasm of large schizonts contained many nuclei, mitochondria, rough and smooth endoplasmic reticulum, and numerous free ribosomes. Refractile bodies were rarely seen in large schizonts. No micronemes or rhoptries were seen in such schizonts. Some nuclei had intra-nuclear spindles and associated spindle poles and centrioles. Such nuclei were considered to be dividing. The formation of blastophores was initiated by an invagination or infolding of the limiting membrane of the schizont. Eventually, this process led to the isolation of spheroidal blastophores, each having a number of nuclei and limited by a single surface membrane derived from that of the schizont. In large schizonts and blastophores, fibrous-appearing bodies were seen. Micronemes appeared to be associated with these bodies. The first indication of merozoite formation was the appearance, just interior to the schizont membrane, of a relatively electron-dense thickening, which was interpreted as the beginning of the inner membrane complex of the pellicle of the merozoite. Merozoite conoids apparently formed de novo in association with this thickening. A nucleus was usually seen in association with such areas, and subpellicular microtubules and micronemes were present at the time the conoid was seen. Such sites of merozoite formation were always seen at a membrane surface. No internal merozoite formation was observed. Merozoite development proceeded by a budding process which consisted of elongation of the merozoite and subsequent incorporation of organelles. After incorporation of the nucleus, micronemes, rhoptry anlagen, mitochondria, Golgi apparatus, and ribosomes, merozoites were separated posteriorly from the schizont by a pinching off process. Merozoites were observed developing at the periphery of blastophores and schizonts, or in association with infoldings of schizonts; in all instances merozoites were formed in close proximity to the limiting membrane of the schizont or blastophore. Mature schizonts had merozoites, residual bodies, and membranous debris. The cytoplasm of host cells became stretched around large schizonts. Such cells were vacuolated in some instances, and were destroyed upon release of mature first-generation merozoites. Merozoites had the organelles characteristically found in sporozoites, except that no refractile bodies were seen. Merozoites had 22 subpellicular microtubules, apparently with a globular substructure. The pellicle of merozoites consisted of an outer unit membrane and an inner membrane complex. The inner membrane comples appeared to consist of a single unit membrane in some specimens and two unit membranes in others.

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