Fine Structure of First-Generation Merozoites of Eimeria bovis

Abstract

The fusiform merozoite is enclosed by a cell membrane. Another membrane underlying the cell membrane encloses the cytoplasm except at the anterior end of the merozoite where this inner membrane terminates forming the polar ring. Approximately 22 subpellicular fibrils extend posteriorly from the polar ring. A conoid, consisting of one or more fibrils wound in a tight helix, is situated within the polar ring. A paired organelle extends posteriorly through the conoid from the anterior end. Each member of the paired organelle is club-shaped having a narrow neck within the conoid region and a wider posterior portion. A median rod parallels the necks of the paired organelle. The region of the merozoite between the conoid and the ovoid glycogen bodies is tightly packed with many tortuous structures having indistinct borders. Numerous ribosomes as well as one or two mitochondria are scattered among these structures. A dense, membrane-enclosed body, possibly a lysosome, is occasionally seen near the mitochondria. The Golgi apparatus lies next to the flattened anterior edge of the nucleus. In some specimens a punctate invagination of the cell surface was seen near the level of the Golgi apparatus. Several cisternae of rough-surfaced endoplasmic reticulum are found both anterior and posterior to the nucleus. The merozoites lie free in a vacuole of the host cell. Blebbing of the host cell's vacuolar membrane releases vesicles into the vacuole. The outer surface of the host cell has numerous microvilli. A fine, fibrous layer exists in the host cell cytoplasm surrounding the vacuole. The cytological characteristics of the firstgeneration merozoites of Eimeria bovis, as revealed by light microscopy, were reported by Hammond, Ernst, and Goldman (1965). The large number of merozoites (about 120,000) contained within the macroscopic host cell and the ease of collection of these host cells provided a simple means for studying the fine structure of this stage of the coccidial life cycle. Electron microscope studies of the merozoites of E. intestinalis (Mossevitch and Cheissin, 1961), E. perforans, and E. stiedae (Scholtyseck and Piekarski, 1965) have been reported. These species differ from E. bovis, however, in the size of the schizont and number of merozoites produced. This study was initiated to provide a detailed description of the ultrastructure of mature first-generation merozoites of E. bovis and the host cell in which they are located. The morphological characters of this stage of E. bovis will be compared with those of the corresponding stages of other species of Eimeria and of other Sporozoa. Received for publication 16 February 1966. * U. S. Department of Health, Education, and Welfare, Public Health Service, NIAID, Laboratory of Parasitic Diseases, Bethesda, Maryland 20014. t Department of Zoology, Agricultural Experiment Station, Utah State University, Logan, Utah. MATERIALS AND METHODS First-generation merozoites of E. bovis were obtained from infected calves as described by Hammond et al. (1965). Immediately after collection, by scraping the intestinal mucosa, the material was placed in a large volume of fixative without washing. Fixation was accomplished by one of three methods. Three per cent glutaraldehyde in Sorensen's phosphate buffer (Sabatine, Bensch, and Barrnett, 1963) was used for some specimens. The material was fixed for 2 hr and then rinsed in buffer with sucrose. This was followed by postfixation in veronal-acetate-buffered Os04 for 2 hr. Some specimens were fixed for 2 hr in veronalacetate-buffered OsO (Caulfield, 1957); others were fixed for a similar period in Dalton's chromeosmium fixative (Dalton, 1955). All fixatives were buffered at pH 7.4 and used at 0 to 4 C. Since it was necessary to mail specimens after fixation from one laboratory to another, they were left for several days in 70% alcohol, or, in the case of glutaraldehyde-fixed material, in the rinsing buffer. All specimens were dehydrated in a graded series of ethyl alcohol. They were then embedded in Epon (Sporn, Wanko, and Dingman, 1962) after being passed through propylene oxide to remove the alcohol. Polymerization was carried out for 16 to 18 hr at 60 C. Sectioning was done with an LKB Ultrotome using a diamond knife. Sections were mounted on bare, 400-mesh grids and stained with lead (Karnovsky, 1961). An RCA EMU-3G electron icroscope operating at 50 kv was used to view and photograph the specimens.