Nuclear Divisions and Refractile-Body Changes in Sporozoites and Schizonts of Eimeria callospermophili in Cultured Cells

Abstract

The nuclear stage and the location and number of refractile bodies in Eimeria callospermophili sporozoites and schizonts were observed at 0.5 to 3-hr intervals from 3 to 20 hr after inoculation of sporozoites into monolayer cultures of Madin-Darby bovine kidney and embryonic bovine kidney and intestine cells. At about 6 hr, the nucleus and nucleolus had changed from a spheroidal to an ellipsoidal shape and had markedly increased in size. The first nuclear division usually occurred 8 to 10 hr after inoculation. Beginning at about 8 hr, the nucleolus became elongated and then assumed a dumbbell shape. At about 9 hr, the nucleolus divided into 2 separate nucleoli and 2 daughter nuclei were formed by infolding of the nuclear membrane between the 2 nucleoli. Between 11 and 15 hr, additional divisions occurred similarly, resulting in 4 to 6 nuclei. In schizonts with more than 6 nuclei, these were small and indistinct. Most freshly excysted sporozoites had a spherical anterior refractile body and a large posterior refractile body. At 6 to 10 hr, the anterior refractile body underwent a decrease in size until it finally disappeared. During this time, granules were formed at the periphery of the anterior refractile body, and these later assumed a random distribution. At 6 to 15 hr, the posterior refractile body decreased in size. The formation of granules at its surface was only rarely observed. After transformation of the sporozoite-shaped schizont to a spheroidal schizont, which occurred about 15 to 20 hr after inoculation, the posterior refractile body usually formed several smaller spherical bodies. In previous work we described the development in cell cultures of first-generation schizonts of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel (Speer, Hammond, and Anderson, 1970). In the course of this work we observed details of nuclear division and changes in refractile bodies in the former species, which are reported herein. MATERIALS AND METHODS Cell lines of embryonic bovine intestine and kidney as well as established cell lines of MadinDarby bovine kidney cells were used in this investigation. The cell monolayers were maintained as described previously (Fayer and Hammond, 1967). Oocysts were also handled as described before (Speer, Hammond, and Anderson, 1970). Three experiments were conducted with each cell type. Cover slips from Leighton tube cultures were examined in double-coverslip preparations (Parker, 1962) with phase-contrast microscopy at 0.5to 3-hr intervals from 3 to 20 hr after inoculation of sporozoites. The observations were made with a Zeiss photomicroscope, 100X neofluar phase-contrast objective, 8x Kpl oculars, and optovar set at 1.25. The appearance of the nucleus or nuclei and Received for publication 9 December 1969. * Supported in part by research grant AI-07488 from the NIAID U. S. Public Health Service. Published as Journal Paper No. 982, Utah Agricultural Experiment Station. the refractile bodies in 80 to 120 intracellular specimens was determined for each time interval in each experiment by examination of living specimens. Measurements were made with an ocular micrometer at a magnification of 1,600X. Each measurement in the following descriptions is in microns and, unless otherwise stated, represents the mean of 30 or more living specimens, with the range in parentheses.