In situ immunocytochemical detection of cells containing antibodies specific for Eimeria tenella antigens

Abstract

A three-step immunocytochemical method for the in situ detection of antibodies specific for Eimeria tenella has been developed. The method is based on the binding of E. tenella antigens to antibodies in cryostat sections of chicken tissues and the recognition of these antigens by rabbit antiserum specific for E. tenella or mouse monoclonal antibodies specific for E. tenella. The rabbit antiserum and mouse monoclonal antibodies were revealed by the immunoperoxidase technique. Suspensions of sonicated sporulated oocysts, incubated with or without various concentrations of the non-ionic detergents Triton X-100 (TX-100) or Nonidet P-40 (NP-40), were used as antigen. Cells containing antibodies specific for E. tenella were detected only when detergent extracts of sonicated sporulated oocysts were used. After chickens were intravenously immunized with a suspension of sonicated sporulated oocyst antigen, cells containing antibodies specific for E. tenella antigens were detected in the red pulp of the spleen. After simultaneous immunoenzyme staining for isotype and antigen specificity, the E. tenella-specific antibody-containing cells were of the IgM isotype after the primary immunization and of the IgM and IgG isotype after the booster immunization. Immune complexes specific for E. tenella on the surfaces of follicular dendritic cells in the germinal centers were also stained. Chickens were also orally infected with sporulated oocysts. In these experiments, cells containing antibodies specific for E. tenella were detected in the lamina propria of the ceca and in the the red pulp of the spleen. Specific immune complexes were also detected in the germinal centers of the cecal tonsils. When detergent extracts of sonicated sporulated oocysts were characterized by immunoblotting, rabbit antiserum specific for E. tenella reacted with proteins ranging in size from 16 kDa to 200 kDa, with major bands of 20 kDa, 24 kDa, 45 kDa, and 100 kDa. Monoclonal antibodies specific for E. tenella recognized only proteins of low molecular weight (20 kDa and 24 kDa) or high molecular weight (80–100 kDa). Immune chicken serum reacted with proteins of low and high molecular weight but especially with proteins of 100 kDa and 113 kDa. This method is the first by which immune complexes and cells containing antibodies specific for parasitic antigens can be detected in situ and may be of value for studies of the local humoral immune response to E. tenella in the mucosa of chickens.