Electron Microscope Observations on the Development of First-Generation Merozoites of Eimeria bovis

Abstract

At the earliest stage observed in the development of the first-generation merozoites of Eimeria bovis the schizont cytoplasm was subdivided into many lobes or spheroidal blastophores; their peripheries being lined by the many nuclei resulting from repeated divisions. The beginning of merozoite production is characterized by the formation of a complex of structures which later comprises the anterior end of the merozoite. A thickened layer forms under the plasma membrane and eventually becomes the inner membrane of the merozoite. Adjacent to a central opening in this layer lies a conoid. Subpellicular fibrils, which radiate from the opening, are closely applied to the inner membrane. As development proceeds, the blastophore membrane is elevated into a cone-shaped projection which later elongates into a fingerlike bud. This bud, the developing merozoite, contains the primordia of the paired organelle, a nucleus with adjacent Golgi apparatus, and other cytoplasmic constituents derived from the blastophore. With further growth of the merozoite, the outer and inner membranes become extended posteriorly; after the early stages, this is associated with an infolding of the membranes into the blastophore. In a late stage of development, the merozoites are completely formed except for an attachment of their posterior ends to the remains of the blastophore. Finally, the attachment is broken, resulting in free merozoites and residual bodies. Eimeria bovis is one of a group of Eimeria species which has unusually large schizonts. Each first-generation schizont of this species produces approximately 120,000 merozoites (Hammond et al., 1946). The literature pertaining to the development of such schizonts was reviewed by Hammond, Ernst, and Miner (1966), in connection with a study of the development of first-generation schizonts of E. bovis with the light microscope. These authors found that in early schizonts the nuclei became arranged in a peripheral layer. This layer then grew inward at a number of places, forming compartments of various sizes. These compartments later gave rise to spherical or ellipsoidal bodies called blastophores, having a single peripheral layer of nuclei. Radial outgrowths of the blastophores formed merozoites, with some cytoplasm remaining as residual bodies. No studies with the electron microscope of schizogony in Eimeria species with large schizonts have been reported. Since the fine structure of the first-generation merozoites of E. bovis is known (Sheffield and Hammond, 1966) the present study was initiated to provide more detailed information concerning the Received for publication 1 March 1967. * Laboratory of Parasitic Diseases, NIAID, NIH, Public Health Service, U. S. Department of Health, Education and Welfare, Bethesda, Maryland 20014. t Department of Zoology, Agricultural Experiment Station, Utah State University, Logan, Utah. cytological events occurring during the development of these merozoites. MATERIALS AND METHODS First-generation schizonts of E. bovis were obtained from calves 12 to 15.5 days after inoculation as described by Hammond, Ernst, and Goldman (1965), except that mucosal scrapings containing schizonts were fixed immediately without prior washing, in order to obtain optimal fixation. The material was fixed in either 3% glutaraldehyde in Sorensen's phosphate buffer (Sabatine, Bensch, and Barrnett, 1963) followed by postfixation in veronal acetate-buffered OsO4, or in Dalton's chrome-osmium fixative (Dalton, 1955). All fixatives were buffered at pH 7.4 and used at 0 to 4 C. Specimens were fixed in Logan then sent to Bethesda in rinsing buffer in the case of glutaraldehyde-fixed materials, or in 70% ethyl alcohol when OsO4 fixative was used. Before the material was shipped the schizonts were concentrated by rotation in petri dishes as previously described (Hammond et al., 1965). With the aid of a dissecting microscope a rough separation of the immature schizonts from the completely mature ones was made by observing degree of opacity and size (immature schizonts were less opaque). All material was dehydrated in a graded series of ethyl alcohol, passed through propylene oxide, and then embedded in Epon according to the procedure of Sporn, Wanko, and Dingman (1962). Polymerization was carried out for 16 to 18 hr at 60 C. Sections were cut with a diamond knife on an LKB Ultrotome. They were mounted on bare, 400mesh grids and stained with lead (Karnovsky, 1961). Photographs were taken with an RCA EMU-3G electron microscope operating at 50 kv.