Date of Award:

5-1-1972

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

James L. Shupe

Committee

LeGrande Ellis

Committee

Paul B. Carter

Abstract

The in vitro development of Eimeria magna was studied using various cell types and compared with corresponding development observed in the intestinal epithelial cells of rabbits at 3, 4 and 5 days after inoculation. Leighton tubes were inoculated with sporozoites and the cultures examined at various intervals after incubation with phase-contrast or interference-contrast microscopy. Mucosal scrapings from infected rabbits were also examined. Mature first- and second-generation schizonts were seen in Madin-Darby bovine kidney (MDBK) cells and epithelioid embryonic bovine liver cells, whereas no development beyond the sporozoite occurred in fibroblastic, embryonic, bovine liver cells, lamb trachea cells and aggregates of primary embryonic bovine kidney cells. Sporozoites increased in size and nuclear divisions occurred to form sporozoiteshaped schizonts. At 40-48 hours, 2-8 nuclei were formed. An average of 41 minutes at 37 C was required for the transformation of sporozoite-shaped schizonts to spheroidal schizonts. Uninucleate first-generation merozoites were apparently formed by a radial budding process which required an average of 56 minutes at 37 C. Mature first-generation schizonts with uninucleate merozoites seen at 3 and 4 days in cell cultures and smears of intestinal epithelial cells, had an average of 12 (4-28) and 17 (4-48) merozoites, respectively. At 72 hours after inoculation, approximately 30 percent of mature first-generation schizonts in MDBK cells had 4 (2-8) multinucleate merozoites, whereas at 3 and 4 days in the rabbit 18 and 23 percent, respectively, of such schizonts had 6 (2-10) multinucleate merozoites. An average of 68 minutes at 37 C was required for complete formation of multinucleate merozoites. Further development of multinucleate merozoites in cell culture was not observed. After entering new host cells, uninucleate merozoites transformed to trophozoites; at 37 C this process required an average of 22 minutes. Some trophozoites gave rise to a pair of second-generation merozoites; during this process, the single nucleus and nucleolus divided and each daughter nucleus with its nucleolus moved into an immature merozoite. Similar pairs of merozoites were seen at 4 and 5 days in ileal epithelial cells in the rabbit. In other specimens, second-generation schizonts with four merozoites were formed after 2 nuclear divisions occurred. Further development of sporozoites beyond second-generation merozoites was not observed. Merozoites of E. magna were obtained from mucosal scrapings taken from a rabbit inoculated 5 1/4 days earlier. These merozoites were inoculated into cultures of Madin-Darby bovine kidney cells, which were then examined with interference-contrast or phase-contrast microscopy at intervals of 1 to 12 hours for 80 hours. Merozoites entered cells and most underwent development into gamonts and oocysts; some formed schizonts. From 12 to 60 hours, young macrogametes gradually enlarged; during this time, the plastic granules increased in size and number. Mature macrogametes averaged 28.5 by 21 µ. From 12-72 hours, microgamonts increased in size, nuclear divisions occurred and, in some, invaginations at the margin of the parasite were present. At 72 and 80 hours, mature microgamonts averaged 32 µ in diameter and had hundreds of microgametes. Extracellular microgametes exhibited slight motility after exposure to a 0.25 percent sodium taurocholate or 2.0 percent bovine bile solution. Fertilization was not observed. Mature oocysts were first seen at 72 hours after inoculation of merozoites.

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