Effects of Monoclonal IgG Antibodies on Eimeria tenella (Coccidia) Sporozoites

Abstract

Although monoclonal antibodies have been generated against certain coccidia (Carosi et al., 1980, Boll. Inst. Sieroter Milan. 59: 25-30; Danforth, 1982, J. Parasitol. 68: 392-397; Sethi et al., 1980, J. Parasitol. 66:192-196), little is known about their effects on various stages of these parasites. In a previous study that used transmission electron microscopy (TEM), we found that 1A, 9D, and 3D3II monoclonal IgG antibodies caused complement-mediated lysis of Eimeria tenella sporozoites (Speer et al., 1983, J. Protozool. 30: 548-554). When viewed by scanning electron microscopy (SEM), we found that sporozoites of E. tenella exposed to these monoclonal antibodies without complement differed considerably in surface texture and size from normal sporozoites. Monoclonal antibodies were obtained as described previously (Kohler and Milstein, 1975, Nature 256: 495-497; Speer et al., 1983, loc. cit.). Sporozoites of E. tenella were incubated for 30 min at room temperature (22 C) with normal mouse ascites fluid or with 1A, 9D, or 3D3II monoclonal IgG antibodies (heat-inactivated) that had been diluted 1: 1 in a Tris-buffered glucose saline solution (pH 7.4) consisting of 10 mM Tris, 10 mM glucose, 170 mM NaCl, and 10% fetal calf serum. Sporozoites were then fixed in 2% glutaraldehyde in Millonig's phosphate buffer (pH 7.2). Sporozoites suspended in Millonig's buffer were captured on Nuclepore filters (0.6 Mm pore size) that had been coated previously with normal mouse serum in order to ensure adherence of the sporozoites. Glutaraldehyde fixative was then pulled through the filter to fix the sporozoites to the filter. Sporozoites on filters were rinsed in buffer, placed in 1% osmium tetroxide for 20 min, rinsed in buffer, dehydrated in ethanol, critical-point dried in a Samdri critical-point dryer, mounted on metal studs, coated with 25 nm of gold: palladium (60:40), and examined with a Zeiss Novascan 30 SEM. All measurements were made at a magnification of X7,000. Significant differences between dimensions of sporozoites exposed to normal ascites fluid and of those exposed to monoclonal antibody were determined by Student's t-test. By using indirect fluorescent antibody assay, lA, 9D, and 3D3II monoclonal IgG antibodies were found to have titers of 1:1.3 X 106, 1:1.2 X 104, and 1:1.6 X 106, respectively. Sporozoites exposed to normal ascites fluid measured 10.01 X 2.02 ,m (9.5-10.5 X 1.8-2.2 Mm; n = 20), whereas those exposed to 9D, 1A, or 3D3II were 9.36 X 2.02 ,um (8-10 X 1.8-2.3 Am; n = 20), 7.97 X 2.18 Am (7-9 X 2-2.5 inm; n = 20), and 6.8 X 2.39 ,m (6-8 X 2.2-2.6 ,im; n = 20), respectively. Sporozoites incubated with 1A and 3D3II were significantly shorter than those incubated with normal ascites fluid (P < 0.0005); 9D caused no significant difference in length (Figs. 1-4). Sporozoites exposed to 1 A and 9D did not differ significantly in width when compared to those exposed to normal ascites fluid, whereas those exposed to 3D3II were significantly wider (P < 0.05). Sporozoites incubated with normal ascites fluid or 9D had relatively smooth surfaces with small protrusions (Figs. 1, 2), whereas those exposed to 1A or 3D3II had rough or wrinkled surfaces with blebs (Figs. 3, 4). Although immune sera have been found to have an immobilizing or lytic effect on sporozoites and merozoites of E. tenella (Bums and Challey, 1965, J. Parasitol. 51: 660-668; Herlich, 1965, J. Parasitol. 51: 847-851), the role that immune sera plays in immunity to this parasite is still unknown. By using SEM, Witlock and Danforth (1982, J. Protozool. 29: 441-445) found that immune chicken serum caused surface bulges, a fibrinous coat and complement-me-