Date of Award:

5-1-1968

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

Merthyr L. Miner

Committee

Raymond T. Sanders

Abstract

Cell line and established cell line cultures were tested for their ability to support in vitro development of Eimeria bovis. Among the cell types tested were embryonic bovine kidney, spleen, thymus, testicle, intestine and trachea cells, embryonic human intestine cells, mouse L cells, and Madin-Darby adult bovine kidney cells. Monolayer cell cultures were serially propagated in disposable prescription bottles and transferred to Leighton tubes for use in experiments. Coverslips from Leighton tube cultures inoculated with sporozoites were examined with phase-contrast or Zeiss Nomarski interference contrast microscopy for as long as 21 days after inoculation. After examination, coverslips were fixed in Schaudinn's fluid, Stieve’s fixative or methanol and stained with iron hematoxylin, Harris' hematoxylin and eosin, Giemsa, bromphenol blue, or PAS-AO. Sporozoites entered cells and underwent development in all cells tested. Multiple infections were seen in all cell types; a maximum of 13 sporozoites were observed in a single Madin-Darby kidney cell. Development proceeded only to the binucleate and trinucleate stages in testicle and L cells, respectively. Mature first-generation schizonts were found only in kidney, spleen, thymus, trachea, human intestine and Madin-Darby kidney cells; a single second-generation binucleate schizont was observed in a trachea cell culture. Generally, the most favorable results were obtained using trachea cells and the next most favorable results with Madin-Darby kidney cells. Sporozoites actively entered cells anterior end first. No passive entrance was observed. A constriction passed backward along the length of the sporozoite as it entered through the host cell membrane. This process required 1 or 2 seconds to over a minute. Sporotoites were also observed leaving cells. Most intracellular sporozoites observed 24 hours or later after inoculation possessed a single posterior refractile body whereas freshly excysted sporozoites usually contained an anterior refractile body also. One or occasionally 2 refractile bodies were observed in the trophozoite and schizont stages. The sequence of changes occurring during transformation from the sporozoite to the trophozoite stage was studied in stained and fresh specimens. The first detectable changes occurred in the nucleus. The peripheral layer of chromatin became thicker, less intensely basophilic and less clearly defined. Simultaneously, the nucleus increased in size and the nucleolus became larger and more eccentric. The sporozoite then assumed the shape of the trophozoite. A crescent-shaped body was often seen at the periphery of the trophozoite and schizont stages in association with the parasite cell membrane or within the surrounding vacuole of the host cell. The significance of this structure is not known. Nuclear and cytoplasmic changes in host cells similar to those noted in histologic sections from infected calves were observed in infected cells. Eight types of media were used to determine the duration of survival of sporozoites in vitro without host cells. Sporozoites were inoculated into each type of medium and incubated at 39 C. Samples, removed at hourly intervals, were examined for the presence of motile sporozoites by phase-contrast microscopy. In medium containing bovine serum, sporozoites retained motility for as long as 21 hours but were motile for only 7 hours in media containing ovine or no serum. Intraoocyst fluid was diluted with saline A and inoculated into Leighton tube cultures of embryonic bovine spleen, thymus and intestine cells to test for the presence of a toxin. All cell types inoculated with the sterile oocyst extract in saline A were killed within a few hours. No pathologic changes were seen in control tubes inoculated with saline A.

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