Development of First‐Generation Schizonts of Eimeria bovis in Cultured Bovine Cells

Abstract

SYNOPSIS. Monolayer primary and secondary cultures of embryonic bovine kidney, spleen, intestinal and testicle cells, and secondary cultures of embryonic bovine thymus, maintained in lactalbumin hydrolysate, Earle's balanced salt solution and ovine serum were observed for a maximum of 21 days after inoculation of E. bovis sporozoites. The sporozoites entered the cells in all of these cultures but underwent development only in primary cultures of kidney and intestinal cells and in secondary cultures of kidney, spleen, thymus, intestinal, and testicle cells. In acellular media, the sporozoites retained motility no longer than 21 hr. In the cell cultures, free motile sporozoites were seen for as long as 18 days after inoculation. Sporozoites entered cells anterior end first; the process of penetration required a few seconds to about a minute. Sporozoites were also observed leaving host cells. Intracellular sporozoites were first seen 3 min after inoculation; they were observed at various intervals up to 18 days after inoculation. In transformation of sporozoites into trophozoites a marked change in size and appearance of the nucleus took place before the change in shape of the body occurred. Trophozoites were first found 7 days after inoculation, multinucleate schizonts after 8 days, and schizonts with merozoites after 14 days. Schizonts containing merozoites were seen only in kidney, spleen, and thymus cells. The mature schizonts were smaller and represented a much lower proportion of the total number than in comparable stages of infections in calves. Schizonts with many nuclei occurred in intestinal cells; the most advanced stage seen in testicle cells was the binucleate schizont. Nuclear and cytoplasmic changes were observed in the infected cells.

Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin–Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells

“…Concerning the selection of host cells and the invasion of cells in vitro however, there was neither host-nor cell type-speci®city, since all cell types oered were seized by the sporozoites. This behaviour in principle was not astonishing, because similar observations had been reported earlier by Fayer and Hammond (1967) and Hammond and Fayer (1968). Even the speed of invasion did not depend on cell source, as sporozoites invaded HUVEC and PUVEC faster than the cattlederived MDBK cells.…”

“…Sporulated oocysts were stored at 4°C and used within 6 months. Sporozoites were excysted from sporulated oocysts and puri®ed by a modi®ed method of Fayer and Hammond (1967). In brief, oocysts were suspended in sterile 0.02 M L-cysteine HCl/0.2 M NaHCO 3 solution and incubated in a 100% CO 2 atmosphere at 37°C for 20 h. Subsequently, the oocysts were resuspended in Hank's balanced salt solution (HBSS; Gibco) containing 0.4% (w/v) trypsin (Sigma) and 8% (v/v) sterile, ®ltered bovine bile (obtained from the local abattoir) and were incubated under microscopic control up to 4 h at 37°C in a 5% CO 2 atmosphere.…”

Fine Structural Aspects of Nuclear Division and Merogony of Eimeria ninakohlyakimovae in Cultured Cells

“…ninakohlyakimovae were collected and prepared for use as described by Kelley and Hammond (1970 lamb trachea cells of the 8th and 13th passages, embryonic lamb thyroid cells of the 15th passage, and embryonic lamb kidney cells of the 22nd passage were obtained as described by Fayer and Hammond (1967). Brockway culture flasks were used for cultivation of the parasite (Roberts, Hammond, and Speer, 1970).…”

Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies

“…Assuming that they are essential for the invasion of cells, this may be the quali®cation for the in vitro behavior of E. bovis sporozoites to leave an occupied cell and invade a new one (Fayer and Hammond 1967; our own unpublished observation). Otherwise, one may speculate that microneme components could have additional, unknown functions in the intracellular life of the parasites.…”