Cytochemical and Cytological Observations on Sarcocystis sp. Propagated in Cell Culture

Abstract

Zoites of Sarcocystis sp., liberated from cysts in the thigh muscles of grackles and inoculated into monolayer cultures of embryonic bovine tracheal cells, entered cells and developed to sexual stages and oocysts. Cytochemical observations were made on all intracellular stages. Neutral lipid droplets were found in the cytoplasm of microgametocytes, macrogametes, and oocysts. Carbohydrates were found as granules in the cytoplasm of zoites, microgametocytes, macrogametes, and some oocysts. Protein was found diffusely or as granules in the cytoplasm and was associated with the nucleus in all stages. Protein-positive granules in macrogametes appeared to take part in the formation of the oocyst wall. Nuclear division in uninucleate and multinucleate microgametocytes was characterized by the formation of a 4-lobed nucleus. DNA occurred as a ring surrounding the nucleus and as granules within the nucleus of zoites, microgametocytes, macrogametes, and oocysts. As microgametes matured, DNA eventually filled the entire nucleus. RNA occurred as single granule in the nucleus of zoites and macrogametes and as 1 or 2 granules in the nuclei of young microgametocytes but was not observed in either microgametes or oocysts. Sexual stages and oocysts of Sarcocystis sp. that develop in cultured cells (Fayer, 1970, 1972) were studied with the electron microscope (Vetterling, Pacheco, and Fayer, 1973), and initial observations regarding cytochemistry were briefly reported (Fayer, 1973). The present study was undertaken to extend those initial observations by studying distribution of lipid, carbohydrate, protein, and nucleic acids. Their distribution was compared with morphological features previously observed with the light and electron microscope in an attempt to correlate chemical composition with structure and thereby gain insight into the function of this parasite's subcellular organelles. MATERIALS AND METHODS Motile zoites of Sarcocystis sp. were liberated from cysts in the thigh muscles of grackles (Quiscalus quiscula) and inoculated into Leighton tubes containing monolayer cultures of embryonic bovine tracheal cells as described by Fayer (1970, 1972). Coverslips were removed from Leighton tubes 1, 6, 24, and 30 hr later. Unless otherwise noted, all staining techniques were taken from Thompson and Hunt (1966). For the study of lipids, coverslip cultures were rapidly frozen on dry ice and stained by the Oil Red O method of Lillie and Ashburn; other cultures were fixed in neutral buffered 10% formalin (NBF) and stained by the Sudan black B method of Chiffelle and Putt; still others were fixed in NBF, incubated in cold acetone (Lillie, 1965), and stained in Received for publication 16 December 1974. * USDA, ARS, Animal Parasitology Institute, Beltsville, Maryland 20705. Sudan black B. For the study of carbohydrates, coverslip cultures were fixed in Carnoy's fluid and stained by the periodic acid-Schiff (PAS) reaction. To test the specificity of the PAS reaction, positive granules were eliminated by acetylation in acetic anhydride in pyridine and restored by deacetylation in alcoholic ammonium hydroxide. The chromic acid-Schiff reaction of Bauer was used to detect glycogenlike polysaccharide after fixation in Carnoy's fluid. Proteins were identified by the bromphenol blue method of Mazia, Brewer, and Alfert (Barka and Anderson, 1965) after monolayers were fixed in Bouin's fixative. Their distribution was confirmed by the method of Himes and Moriber (Humason, 1962) by using Naphthal yellow S after fixation in Carnoy's fluid. For detection of nucleic acids, all monolayers were fixed in Carnoy's fluid. Control preparations were then incubated (37 C, 4 hr) in Tris buffer (pH 7.5) alone, whereas others were incubated in Tris buffer containing deoxyribonuclease (DNase) or ribonuclease (RNase). Loss of affinity for gallocyanin-chromalum (G-C) stain of Einarson by host cell nuclei and nucleoli indicated DNase and RNase activity. The distribution of DNA was confirmed by the method of Himes and Moriber (Humason, 1962), in which the Feulgen reaction is used.