Penetration of Eimeria larimerensis Sporozoites into Cultured Cells as Observed with the Light and Electron Microscopes

Cell cultures of Madin-Darby bovine kidney (MDBK) were used to study with the light and electron microscopes the process of penetration of cells by Eimeria larimerensis sporozoites. The fine structure of these sporozoites was also studied. Monolayers from Leighton tubes were covered with a concentrated suspension of sporozoites and immediately observed in double-coverslip preparations with phase-contrast microscopy. For electron microscope study, sporozoites were added to and mixed well with a suspension of MDBK cells, centrifuged for 4 min, fixed immediately, and prepared for study with the electron microscope. Frequently, the Golgi complex of the sporozoites was located in an indentation of the nucleus and partially surrounded by a fold of the nuclear envelope. The inner layer of the pellicle consisted of 2 unit membranes. Wavelike elevations of the pellicle were seen; these may be involved in locomotion. An intranuclear inclusion, consisting of microtubulelike fibrils, was observed. Vacuoles with particulate matter similar to that of the central vacuoles of the Golgi complex and with extensions running anteriorly into the conoid area were seen in extracellular sporozoites and in sporozoites entering cells. The bodies of sporozoites were usually constricted as they entered and left host cells and host cell nuclei; this was also observed in sporozoites moving through the cytoplasm of the host cell. During penetration, the host cell membrane was either interrupted at the initial site of entry or was interrupted after becoming invaginated for a short distance. Escape of host cell cytoplasm occurred frequently after sporozoites left host cells, but only seldom after entrance. Some intracellular sporozoites fixed 4 min or less after inoculation were surrounded by a host cell membrane; others were not. Some sporozoites which were fixed in the process of leaving host cells had a thin layer of host cell cytoplasm covering the portion of the body which was outside of the host cell, and some extracellular sporozoites with such a covering were seen. The process of host cell penetration by sporozoites of several Eimeria species has been studied in vitro with the light microscope. E. larimerensis is especially favorable for such a study because of the relatively large size of its sporozoites and because they enter cultured cells readily (Speer and Hammond, 1970). A detailed study with the light and electron microscopes of sporozoite penetration into cultured cells, as well as the fine structure of extraand intracellular sporozoites of E. larimerensis, is reported herein. MATERIALS AND METHODS Oocysts of Eimeria larimerensis were collected from experimentally infected ground squirrels (Spermophilus armatus), cleaned, sporulated, and sterilized as described previously (Speer, Hammond, and Anderson, 1970). They were then Received for publication 15 September 1970. * Supported in part by research grant AI-07488 from the NIAID, U. S. Public Health Service, and by Public Health Service Fellowship 1-F01GM44456-01 from the Institute of General Medical Sciences. Published as Journal Paper No. 1080, Utah Agricultural Experiment Station. incubated for 15 min in an excysting medium consisting of 0.25%o trypsin and 0.75% sodium taurocholate in saline A. The free sporozoites were washed and resuspended in minimum essential medium (MEM). For studying penetration, 2day-old cultures of Madin-Darby bovine kidney (MDBK) cells in Leighton tubes or 8-oz Brockway culture flasks were used. In the light microscope study, cover slips with monolayers were removed from Leighton tubes, covered with a few drops of suspended sporozoites (0.8 to 1 million sporozoites/ml), and immediately observed in doublecoverslip preparations (Parker, 1962). In the electron microscope study, the cell monolayer in an 8-oz Brockway culture flask was harvested with a trypsin-versene solution. The suspension was centrifuged and the pellet was resuspended in 3 ml of MEM. Five million sporozoites in 3 ml of MEM we added to the tube containing suspended MDBK cells. These were mixed well and immedi tely centrifuged at 750 g for 4 min. The fixative was added immediately after removing the supernatant. Some pellets were fixed according to the method described by Karnovsky (1965), using cacodylate buffer. Other pellets were fixed with 2.67% glutaraldehyde in cacodylate buffer for 1 hr, washed with buffer for 1/2 hr, and postfixed in 2.5% osmium tetroxide in cacodylate buffer for 1 hr. The fixed cells were dehydrated in 35 and 50% ethanol for 10 min each and stained with 1% uranyl acetate and 1% phosphotungstic