Antigens of In vitro-Produced First-Generation Merozoites of Eimeria bovis (Apicomplexa)

Abstract

Production of mammalian coccidia in cell culture and subsequent use of in vitro-produced organisms as a source of antigen has been mainly restricted to Toxoplasma gondii (Chernin and Weller, 1957, Journal of Parasitology 43: 33-39; Krahenbuhl et al., 1971, Journal of Parasitology 57: 386-390; Braveny et al., 1978, Tropenmedizin Parasitologie 29: 432-434). We describe here a procedure for the production in vitro of large numbers of first-generation merozoites of Eimeria bovis. Also presented are data concerned with the antigenic similarities of merozoites of E. bovis obtained in vivo or in vitro. Oocysts of E. bovis were collected from the feces of experimentally infected Holstein-Friesian bull calves, sporulated, purified and cleaned as described previously (Fayer and Hammond, 1967, Journal of Protozoology 14: 764-772). Oocysts were stored at 4 C in aqueous 2.5% (w/ v) K2Cr207 for no more than 6 mo prior to use. Sporozoites were excysted from oocysts and separated from oocyst and sporocyst debris immediately before inoculation of cultured cells for in vitro production of first-generation merozoites (Fayer and Hammond, loc. cit.; Speer, 1983, The coccidia in in vitro cultivation of protozoan parasites, J. B. Jensen (ed.). CRC Press, Inc., Boca Raton, Florida, 297 p.; Larsen et al., 1984, Journal of Parasitology 70: 597-601). In vivo-produced first-generation merozoites were obtained from an experimentally infected calf as described previously (Reduker and Speer, 1985, Canadian Journal of Zoology 63: 2478-2480). An established cell line of bovine monocytes (BM) was used as a source of host cells for cultivation of first-generation merozoites. The source and characteristics of this cell line, culture medium (CM), and the ability of BM to support penetration and development of E. bovis have been described previously (Speer et al., 1985, Infection and Immunity 50: 566-571). Each of four 150-cm2 polystyrene Corning tissue culture flasks was inoculated with enough BM to produce a 75% confluent monolayer within 24 to 48 hr. Each flask was then inoculated with 40 ml of CM containing 7.5 x 105 sporozoites, which resulted in an inoculum density of 104 sporozoites/ cm2 BM. Culture flasks were incubated at 38 C in 5% CO2-95% air in a Forma Scientific continuous flow CO2 incubator. When mature meronts and extracellular merozoites were detected with phase-contrast microscopy, the CM containing free merozoites was removed daily and replaced with fresh CM. Merozoites were harvested from each flask at 1021 days after sporozoite inoculation (DAI). After the culture flask was gently rapped 20 x with the palm of the hand, the medium was rocked back and forth 20 x, then decanted into a sterile 50ml centrifuge tube. Ten ml Hanks' balanced salts solution (calcium and magnesium free, pH 7.4; HBSS) were added to each flask and the process was repeated, after which fresh CM was added to each flask. The harvested suspension, which contained merozoites and some host cells, was pelleted by centrifugation at 200 g for 10 min, resuspended in 2-3 ml HBSS, and then agitated with 8-10 strokes on a motor-driven teflon-coated tissue grinder in order to disrupt any intact mature meronts. Numbers of merozoites harvested each day from each flask were estimated by counting on a hemacytometer, and mean number of merozoites harvested (?+ 1 SD) was calculated. At 21 DAI, the width of each flask was examined by phase-contrast microscopy (x 200) for number of intracellular sporozoites, meronts (immature and mature) and degenerate or ruptured meronts. The relative frequency of each stage was calculated as a percentage of total parasite stages observed. After examination, BM and parasites were removed from each flask (0.35% EDTA in RPMI 1640, 38 C, 15 min), and numbers of merozoites were determined as outlined above. Merozoites obtained from cultures were combined, centrifuged, and disrupted in 2% sodium dodecyl sulfate (SDS), 10% glycerol, 6.25 x 10-2 M Tris (hydroxymethyl) aminomethane, 4%