Harley G. Sheffield

Toxoplasma gondii: The Oocyst, Sporozoite, and Infection of Cultured Cells

The infective form of Toxoplasma gondii fouind in cat feces is an oocyst wihich, when sporutlated, resembles that of the genus Isospora in havinig two sporocysts. Sporozoites obtained by artificial excystation of the oocyst are infective for monkey kidney cell cultures. Ultrastructural characteristics of sporozoites resemble those seen previously in proliferative stages of Toxoplasma gondii.

Fine structure of first-generation merozoites of Eimeria bovis.

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 cells 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 Sorensens 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 Daltons 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.

Electron microscope observations on the development of first-generation merozoites of Eimeria bovis.

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 Sorensens phosphate buffer (Sabatine, Bensch, and Barrnett, 1963) followed by postfixation in veronal acetate-buffered OsO4, or in Daltons 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.

Toxoplasma gondii: transmission through feces in absence of Toxocara cati eggs.

When incubated at room temperature (23�C) for 3 to 14 days, feces from cats infected 4 to 8 days with Toxoplasma gondii, and free of Toxocara cati eggs, produced toxoplasmosis in mice. Results indicate that the nematode egg is not necessary for transmission of the parasite.

Effect of pyrimethamine and sulfadiazine on the fine structure and multiplication of Toxoplasma gondii in cell cultures.

Rhesus monkey kidney cell cultures were inoculated with Toxoplasma gondii organisms obtained from peritoneal fluid of mice infected with the RH strain. Pyrimethamine and sulfadiazine were added either singly or in combination to the cultures 4 hr after inoculation. Twenty-four hours later the effect of the drugs on the parasites were studied by light and electron microscopy. Pyrimethamine (1.0 mug/ml) inhibited multiplication of the parasites and caused striking morphological changes. Organisms were rounded and often had a fragmented nucleus. Division was inhibited as indicated by abnormal daughter membrane formation during endodyogeny. No effect was evident in sulfadiazine-treated parasites when concentrations up to 50 mug/ml were used. However, combination of ineffective levels of pyrimethamine (0.1 mug/ml) and sulfadiazine (0.5 mug/ml) produced effects similar to those seen at a higher concentration of pyrimethamine indicating a synergistic action of the 2 drugs.

Ultrastructure of the cyst of Sarcocystis muris.

Cysts of Sarcocystis muris develop within muscle cells and each is bounded by a parasitophorous vacuole membrane. Closely spaced spherical blebs formed from this membrane extend into the muscle cell cytoplasm. A dense substance fills the cavity of the bleb and occupies the vacuolar space immediately adjacent to the membrane. The remainder of the vacuole is filled with a moderately dense matrix within which the parasites develop. At 40 days after infection only metrocytes are present, characterized by their ovoid shape, lightly stained cytoplasm, amylopectin-like granules, and lack of micronemes. Metrocytes divide by a process resembling endodyogeny and eventually produce bradyzoites. By 78 days after infection, at which time the cyst is infective for cats, the few remaining metrocytes are located at the cyst periphery but most organisms are elongated and contain organalles characteristic for bradyzoites including micronemes, dense granules, and amylopectin. Structures indicative of division were not seen in bradyzoites. Rhoptries are few in number. Numerous vesicles of smooth endoplasmic reticulum accumulate in the cytoplasm of muscle cells adjacent to the periphery of the enlarging cyst but significant destruction of muscle fibers containing cysts with viable organisms was not seen in specimens fixed between 40 and 325 days after infection. Unusual lamellar structures were seen in some parasitized muscle cells and intracystic tubules occurred in some cysts.

Experimental studies on Trichuris muris in mice with an appraisal of its use for evaluating anthelmintics.

Attempts to infect white mice from five commercial sources with Trichuris muris yielded discouraging results. DBA-2 hybrid mice, particularly weanlings, were the most useful experimental hosts among nine types of rodents studied for susceptibility. In these mice, from 10 to 27 per cent of the administered eggs developed. Approximately 1 month was required for the worms to mature, and mature worms persisted for 2 to 3 months. However, the infections appeared to be of limited value in drug screening for trichuricidal activity, since they were refractory to treatment with the known trichuricides: dithiazanine iodide and pyrvinium chloride. Infection of laboratory rodents with Trichuris muris (Schrank, 1788) was first reported by Shikhobalova (1937). He experimentally infected white mice to study the developmental stages, with reference to the use of the worm for chemotherapeutic testing. Tissue reactions to the parasite in the cecum of white mice were described by Efremov and Shikhobalova (1939), and resistance of white mice to superinfection with T. muris was demonstrated by Shikhobalova (1940, 1941). Attempts to induce immunity in mice by oral or parenteral vaccination with extracts of T. muris were reported by Leikina (1944). Previously, T. vulpis in dogs had been the main experimental trichurid infection (Lammler, 1958). The limitations of work with this host, in contrast to a small rodent, are readily apparent. Fahmy (1954) induced T. muris infections in laboratory-reared albino mice with eggs from wild house mice. He described the main points of the life cycle, including the morphogenesis of the larval stages, but failed to disclose the strain of mice used, the proportion of exposed mice that became infected, or the relative susceptibility among strains. Each of these factors may influence decisively the maintenance of the infection on a routine basis in the

Activity of the anticoccidial compound, lasalocid, against Toxoplasma gondii in cultured cells.

The activity of the anticoccidial drug, lasalocid, was tested against Toxoplasma gondii in cell cultures. Multiplication of parasites was inhibited by 0.05 mug/ml of lasalocid added to the cultures prior to adding the parasite inoculum, with the parasite inoculum, or after the parasites had penetrated the culture cells. Penetration of culture cells was inhibited when 0.05 mug/ml lasalocid was added with the parasite inoculum. Incubation of extracellular parasites in 0.5 mug/ml lasalocid had no effect on penetration or multiplication. Ormetoprim, sulfadimethoxine, and a combination of the 2 were less effective than lasalocid. Monensin exhibited an inhibitory effect in all experiments.

The effects of reference anthelmintics against Nematospiroides dubius and oxyurids in mice relative to screening procedures for new drugs.

This paper deals with a continuing search for adequate screening procedures that would expedite the development of better anthelmintics, particularly for combatting intestinal nematodes. Reflection upon the general problem and a review of the literature attest so clearly to the inadequacy of available methods as to require no elaboration upon the need for better methods. Nematospiroides dubius Baylis, 1926, has been demonstrated to be infectious for laboratory mice (Baker, 1954). This contribution along with the oxyurids Aspiculuris tetraptera Schulz, 1924, and Syphacia obvelata Seurat, 1916, which occur as cosmopolitan natural infections, enables simultaneous study of drugs in mice against three intestinal nematodes that not only represent different taxonomic groups but also have predilections for different portions of the intestinal tract. N. dubius resides in the upper portion of the small intestine (unpublished observations) while S. obvelata and A. tetraptera have predilections, respectively, for the cecum and large intestine (Thompson and Reinertson, 1952). The effects of selected anthelmintics under various treatment regimens against these infections constitute the body of this report.

Fine structure of immature cysts of Sarcocystis cruzi.

Sarcocystis cruzi forms cysts in striated muscle of the bovine host following schizogony. The fine structure of the immature cyst within muscle fibers of the ventricular myocardium was studied in relation to its development and to the multiplication of parasites within it. The young cyst is enclosed by a cyst wall containing numerous small protuberances. Metrocytes within the cyst are irregular in shape and are separated from each other and the cyst wall by a thin layer of ground substance. The parasite multiplies by endodyogeny within the metrocyte. As the cyst enlarges, the host muscle fiber is disrupted and large protrusions are present in the cyst wall.