title: Eimeria maxima TRAP family protein EmTFP250: subcellular localisation and induction of immune responses by immunisation with a recombinant C-terminal derivative.
abstract: EmTFP250 is a high molecular mass, asexual stage antigen from Eimeria maxima strongly associated with maternally derived immunity to this protozoan parasite in hatchling chickens. Cloning and sequence analysis has predicted the antigen to be a novel member of the thrombospondin-related anonymous protein (TRAP) family of apicomplexan parasites. Members of the TRAP family are microneme proteins and are associated with host cell invasion and apicomplexan gliding motility. In order to assess the immunogenicity of EmTFP250, a C-terminal derivative encoding a low complex, hydrophilic region and putative transmembrane domain/cytosolic tail was expressed in a bacterial host system. The recombinant protein was used to immunise mice and chickens and found to induce strong IgG responses in both animal models as determined by specific ELISAs. Using Western blotting, protective maternal IgG antibodies previously shown to recognise native EmTFP250 recognised the recombinant protein and, in addition, antibodies raised against the recombinant protein were shown to recognise native EmTFP250. Localisation studies employing immuno-light microscopy and immuno-electron microscopy showed that antibodies to the recombinant protein specifically labeled micronemes within merozoites of E. maxima. Furthermore, antibodies to the recombinant EmTFP250 derivative showed similar labeling of micronemes within merozoites of Eimeria tenella. This study is further suggestive of a functional importance for EmTFP250 and underscores its potential as a candidate for a recombinant vaccine targeting coccidiosis in chickens.
authors.affiliation: Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Westbourne St, Gore Hill, Sydney, NSW 2065, Australia. david.witcombe@uts.edu.au
authors.forename: David M
authors.initials: DM
authors.lastname: Witcombe
authors.forename: David J P
authors.initials: DJ
authors.lastname: Ferguson
authors.forename: Sabina I
authors.initials: SI
authors.lastname: Belli
authors.forename: Michael G
authors.initials: MG
authors.lastname: Wallach
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000032:analysis / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis / Q000276:immunology*; D002645:Chickens / Q000276:immunology; D003001:Cloning, Molecular; D004539:Eimeria / Q000276:immunology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D007114:Immunization / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology; D007421:Intestine, Small / Q000276:immunology; D051379:Mice; D016253:Microscopy, Immunoelectron / Q000379:methods; D015800:Protozoan Proteins / Q000032:analysis / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D019699:Thrombospondins / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019699:Thrombospondins