Development of the First-Generation Schizonts of Eimeria auburnensis

Thirty-one Holstein-Friesian calves were inoculated with Eimeria auburnensis oocysts; one or more were killed at daily intervals from 4 to 17 days after inoculation. Also, eight calves were infected by the introduction of excysted sporozoites of E. auburnensis into intestinal fistulas, and two calves by placing sporozoites into the intestine via laparotomy. The tissues from calves were prepared by several histological techniques for microscopic examination. First-generation asexual stages were located in epithelial cells at or near the base of the crypts in the jejunum and ileum. Numerous intracellular sporozoites were observed in fistulas of calves killed 4 and 5 days after inoculation. No typically rounded, uninucleate trophozoites were found. In 5-day fistulas, many of the parasites had an appearance similar to that of sporozoites, except that they were larger and had two to six nuclei. Often, such sporozoite-shaped schizonts had lateral bulges with one or more nuclei. This probably represents a stage in the formation of spheroidal schizonts. Early schizonts had nuclei which were randomly arranged; in later schizonts the nuclei assumed a peripheral location. Beginning on the 8th day after inoculation, numerous invaginations of the peripheral nuclear layer of the developing schizont occurred. Each invagination had a lightly staining lumen, which was continuous with the parasitophorous vacuole; each wall bordering the lumen had a single layer of closely spaced nuclei. In schizonts approaching maturity, merozoites were formed by budding from the walls of the invaginations. In mature schizonts, first observed at 10 days after inoculation, the merozoites were randomly distributed throughout the space occupied by the schizont, including the parasitophorous vacuole. Such schizonts contained many thousands of merozoites and occupied the whole width of the crypt, with few or no epithelial cells remaining along the sides. A small amount of glycogen was observed in intracellular sporozoites; more occurred in schizonts, and first-generation merozoites had many granules of glycogen in the middle two-thirds of their bodies. Nuclei of asexual stages were Feulgen-positive. Crescent-shaped bodies of unknown function were associated with 5-day sporozoites and 5and 6-day schizonts. In response to the presence of the parasite, host cell nuclei increased in size; the chromatin became less prominent, and the nucleoli became greatly enlarged. There was no apparent increase in volume of the host cell cytoplasm. Some of the epithelial cells adjacent to the parasitized cells underwent degeneration. Eimeria auburnensis Christensen and Porter, 1939 is a coccidium of cattle having a low degree of pathogenicity as compared with E. bovis and E. zuernii. E. auburnensis is of special interest because the sexual stages are located more deeply in the host tissues than usual, parasitizing cells of the lamina propria, rather than epithelial cells (Hammond, Clark, and Miner, 1961). Little is known about the location and development of the asexual stages in the life cycle of this species. Davis and Bowman (1962) found large schizonts and microgametocytes in the lamina propria near the muscularis mucosae in the small intestine Received for publication 25 October 1968. * Supported in part by NSF Research grant GB5338, and by Public Health Service Fellowship 5-F1-GM-31,285 from the Institute of General Medical Sciences. Published as Journal Paper No. 831, Utah Agricultural Experiment Station. tPresent address: Department of Biology, Andrews University, Berrien Springs, Michigan 49104. of two calves. The relatively large size of the sporozoites (Nyberg and Hammond, 1965; Hammond, Chobotar, and Ernst, 1968) provides a favorable situation for the study of early postinvasion changes in the developing parasites and in host cells. The study reported herein was undertaken to determine the development of the first-generation schizont of E. auburnensis and its relationship to the host cells and tissues. METHODS OF PROCEDURE All calves were purchased locally when less than 1 week old, and housed in individual 5by 10-ft pens constructed of plywood panels. The rear half of each pen was covered with corrugated sheet metal. The floor consisted of a 6-inch layer of coarse gravel, covered with straw, which was changed weekly. At the time of purchase, each calf was given intramuscularly 500,000 international units of vitamin A, 75,000 of vitamin D2, and 50 of vitamin E. Each calf was fed twice daily 2 to 3 pints of raw whole milk mixed with 1 pint of warm water; also, hay and mixed grain were provided. At the beginning of the 4th or 5th week the animals were placed on a diet of