Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine-and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria. Worldwide economic losses due to these parasites have been estimated to exceed 1.2 billion U.S. dollars per annum (41). The most virulent species is Eimeria tenella, causing severe hemorrhagic enteritis by infection of the epithelium and submucosa of the ceca and, eventually, death of infected chickens (24).Eimeria infections occur by ingestion of oocysts (24). In the intestine, oocysts release four sporocysts, each containing two sporozoites. After excystation, motile infective sporozoites actively enter cells in the epithelium of the cecum. Three rounds of asexual multiplication in the epithelium and submucosa are then followed by differentiation to sexual stages of micro-and macrogametocytes (23). After fertilization of macrogametes, a complex, two-layered wall is secreted around the young oocyst by exocytosis of wall-forming body type I and type II (WFBI and WFBII) (35). While the 10-nm-thick outer oocyst wall is built up by the contents of WFBI, the 90-nm inner oocyst wall is composed mainly of glycoproteins that were stored in WFBII (31, 37). The oocyst displays a remarkable rigidity and protects the parasite from several physically and chemically adverse influences, such as commonly used disinfectants (34). A potential use of gametocyte antigens involved in formation of the oocyst wall as protective transmission-blocking vaccines has been described for Eimeria maxima (2,4,25,(38)(39)(40)46).The formation of oocyst and sporocyst walls and sporozoite excystation are rather complex processes that we are just beginning to underst...

Two monoclonal antibodies (mAbs) raised against the macrogamonts of Eimeria tenella identified antigens located in the wall-forming bodies of type I (WF I) and type II (WF II) by indirect immunofluorescence and by immunoelectron microscopy. With these mAbs, the involvement of both types of wall-forming body at the protein level in the formation of the inner and outer oocyst walls of E. tenella was shown by indirect immunofluorescence assay. On Western blots of pure macrogamont, mAb E1D8 against WF I reacted with a series of bands between 42 kDa and 105 kDa. In pure, unsporulated extract, this mAb recognized a complex of bands between 26 kDa and 153 kDa. mAb E2E5 against WF II, on Western blots of pure extract of macrogamonts, recognized an antigen of 51 kDa. Later in the development, after the formation of the inner oocyst wall, mAb E2E5 reacted with three polypeptide of 23, 25 and 30 kDa. Proteolytic processing may be forwarded as the mechanism regulating the distinct regulation protein involved in the oocyst wall.

Eimeria tenella is the causative agent of caecal coccidiosis in chicken. Infection of chicken is initiated by ingestion of sporulated oocysts. The oocyst wall has two typical layers: a smooth outer layer formed by wallforming body I (WF I) and an inner layer originating from WF II. We observed that the inner layer is circumscribed by a hitherto undocumented linear suture and propose its role in the process of infection.