title: Growth of Eimeria alabamensis from Cattle In Vitro and the Changes in Fine Structure Associated with Schizogony
Creators: name: Sampson, J. Robert
Publisher: name: Utah State University
Year: 1970

description: Monolayer primary cultures of cells from bovine embryonic intestine (BEint), kidney (BEK), spleen (BES), and thyroid (BET) and cell line cultures of embryonic bovine trachea ( EBTr) and synovium (BESy)as well as established cell line cultures of Madin-Darby bovine kidney (MDBK), human intestine (Int 407), and Syrian hamster kidney (BHK-21) were inoculated with freshly excysted sporozoites of Eimeria alabamensis and observed for 4 days. Sporozoites penetrated all cell types during the first 24 hours in culture. Numerous intracellular sporozoites, trophozoites, and binucleate schizonts were seen in all cell cultures except Int 407. The best development occurred in BES and MDBK cells. Mature schizonts were first found at 2 days after inoculation in all cell types except for BHK-21 and Int 407 cells in which they were first observed at 3 days. Large, 14.2 microns (11-18-5) by 10.2 microns (8.5-11), schizonts with 6-15 short, stubby merozoites, each with two refractile bodies, were found at 2 and 3 days in all cells except BESy, Int 407, and BHK-21. Small, 9.7 microns (5.5-13) by 6 microns (5-8.5), highly refractile, compact schizonts with 6-10 long, slender merozoites, each with two refractile bodies, were found at 3 days after inoculation in all cell types. All merozoites had left the schizont by the end of the third day in culture; few parasites were seen thereafter. Intracellular merozoites were found at 4 days, Some second-generation trophozoites were seen in host cell nuclei. Development within the host cell nucleus, the normal development site in the host animal, was observed infrequently in cell cultures. Intranuclear sporozoites were not seen until 2 days after inoculation. Thereafter, developmental stages similar to those occurring in the cytoplasm were observed in the nucleus. Small intranuclear schizonts, having 6-10 merozoites each with two refractile bodies, were found at 3 days.
Freshly excysted sporozoites and intracellular stages, found from 5 minutes to 72 hours after inoculation of sporozoites into cultured cells, were studied with the electron microscope.
Extracellular sporozoites were similar to intracellular sporozoites observed 5 minutes after inoculation, The numerous polysaccharide granules present in these disappeared during the first day of intracellular existence, Some sporozoites transformed into trophozoites which ultimately lost the inner membrane and anterior organelles; others retained these organelles and became sporozoite-shaped schizonts with 2-4 nuclei. Centrioles, each with nine peripheral tubules and one central tubule, were observed in association with intranuclear spindle fibers in dividing nuclei within trophozoites or schizonts. The sporozoite-shaped schizonts later transformed into spheroidal schizonts which gradually lost the inner membrane and anterior organelles.
Merozoite formation occurred in two ways. In sporozoite-shaped schizonts, anlagen of merozoites appeared in the interior of the cell. Extensions of the inner membrane of the parent cell penetrated deep into the cytoplasm. Such internal membranes evidently participated in isolating the immature merozoite from the cytoplasm of the schizont, along with infoldings of the surface membrane of the schizont. Merozoites formed in this manner were long and slender.
Spheroidal schizonts formed by trophozoites usually had a single surface membrane with inner membrane present only in various small areas about the periphery of the cell. Conoids were found beneath the surface membrane and adjacent to the nucleus. In later stages, developing merozoites grew outward from the surface of the schizont, and infoldings of the limiting membrane of the schizont occurred at the margins of each. Such merozoites appeared short and stubby.