Eimeria tenella microneme protein EtMIC3: identification, localisation and role in host cell infection.

The gene coding for Eimeria tenella protein EtMIC3 was cloned by screening a sporozoite cDNA library with two independent monoclonal antibodies raised against the oocyst stage. The deduced sequence of EtMIC3 is 988 amino acids long. The protein presents seven repeats in tandem, with four highly conserved internal repeats and three more divergent external repeats. Each repeat is characterised by a tyrosine kinase phosphorylation site, WRCY, and a reminiscent motif of the thrombospondin1 (TSP1)-type I domain, CXXXCG. The protein EtMIC3 is localised at the apex of free parasite stages. It is not detected in the early intracellular parasite stage but is synthesised in mature schizonts. Secretion of the protein is induced when sporozoites are incubated in complete medium at 41 degrees C. Strangely enough, the two independent mAb that allow cloning of EtMIC3 interfere with parasitic growth in different ways. One is able to inhibit parasite invasion whereas the other inhibits development. Expression and localisation of the protein EtMIC3 are consistent with a protein involved in the invasion process as is expected for a microneme protein.

Aspartyl proteinase genes from apicomplexan parasites: evidence for evolution of the gene structure.

Aspartyl proteinases are a widely distributed family of enzymes. All vertebrate aspartyl proteinases share a conserved nine-exon gene structure, but in other organisms the structure of aspartyl proteinase genes varies considerably. The exon-intron patterns generally reflect phylogeny based on amino acid sequences. However, close comparison of these gene structures reveals some striking features, such as the conservation of intron positions and intron phases between aspartyl proteinases from nematodes and apicomplexans. Here, we discuss the implications of gene structure for the possible evolution of the aspartyl proteinase family, with particular reference to the plasmepsins of Plasmodium falciparum and eimepsin from Eimeria tenella.

Genomic organisation and developmentally regulated expression of an apicomplexan aspartyl proteinase.

The cDNA for an aspartyl proteinase, termed eimepsin, was isolated from an Eimeria tenella sporulated oocyst library and the deduced amino acid sequence found to be almost identical to a previously described aspartyl proteinase from E. acervulina (97.4% amino acid identity). An E. tenella cosmid clone covering the entire eimepsin gene was cloned and characterised. Sequencing revealed that the eimepsin gene spans 2.9 kb and consists of 18 exons and 17 introns. The 5' flanking region sequence of the gene contains a putative transcriptional promoter sequence (TATAAA box) and three potential transcription initiator sites (Inr sites). Expression of eimepsin at the mRNA and protein level is developmentally regulated during oocyst sporulation. The eimepsin transcript was detected in unsporulated oocysts and increased in abundance during the early part of sporulation when the oocyst undergoes nuclear division and blast formation. Thereafter, the level of the eimepsin transcript decreases and in the excysted sporozoite, no eimepsin-specific RNA was detected. Expression of eimepsin lags behind transcript expression by some hours, and the protein accumulates in the oocyst during sporocyst and sporozoite formation.

Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process.

Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.

Adjuvant effect of cholera toxin on systemic and mucosal immune responses in chickens infected with E. tenella or given recombinant parasitic antigen per os.

We investigated the adjuvant effect of cholera toxin (CT) on the intestinal and systemic immune systems of chickens. The CT was given orally, mixed with a non-replicating antigen, a recombinant Eimeria protein, IPEI, or with a replicating one, Eimeria tenella parasite. There were increases in the specific IgA and IgG responses to the recombinant protein IPEI, with a significant anti-1PE1 IgG response in the duodenum (p < 0.05) and caecum (p < 0.05) 4 weeks after immunization and a specific IgA (p < 0.05) response in the duodenum after 3 weeks. A transient anti-1PE1 IgG (p < 0.05) response was detected in the serum 1 week post-injection and an IgA response (p < 0.05) at 2 weeks. CT given with the replicative parasite caused no change in the intestinal and systemic immune responses with 1 or 3 immunizations although a specific antiparasitic in vitro proliferation of the spleen cells from infected chickens was observed. Nevertheless, 0.05 mg CT given per os to chickens was strongly immunogenic in both experiments. A strong serum IgG (p < 0.01) response was detected as soon as 1 week after the end of the immunization protocol with 1PE1 and 2 weeks after infection with E. tenella. Strong anti-CT IgG responses were also detected by the second week post-immunization in the duodenum and caeca (p < 0.01). Hence, CT can be used as a mucosal adjuvant in chickens to improve the intestinal immune response.

Purification of first-generation Eimeria tenella schizonts.

A rapid and simple method for purifying first-generation Eimeria tenella schizonts was developed with infected chicken cecal tissue. The schizonts were harvested from the tissue by treatment with a mixture of 3 enzymes: hyaluronidase, dispase, and collagenase. Subsequent purification of the parasites by filtration through gauze and centrifugation resulted in a clean final preparation compared with the starting material. Microscopical and biochemical examinations, performed to assess the quality of the schizont purification, showed a high degree of purity. This procedure may be used to obtain clean schizonts minimally contaminated with host-cell debris in order to study this parasitic stage.

Purification of a leucine aminopeptidase from Eimeria falciformis.

A leucine aminopeptidase was purified from the oocysts of Eimeria falciformis using affinity chromatography and gel filtration techniques. It had a molecular weight of 45-50 kDa. Its maximal activity against leucyl-p-nitro anilide was at pH 8.6. It is a metallo-enzyme highly inhibited by bestatin.

Comparison of outbred lines of chickens for resistance to experimental infection with coccidiosis (Eimeria tenella).

Coccidiosis causes dramatic economic losses in the poultry industry. Next to the extensive use of anticoccidial drugs, improving genetic resistance of birds to this parasitic disease represents an attractive alternative. An experiment was run in order to identify lines of chickens resistant and susceptible to coccidiosis as a tool to search for genetic markers of resistance. Five outbred lines were used: two Egyptian lines (Mandarah and Fayoumi), a Rhode Island Red line, and two White Leghorn lines (WLB21 and WLDW). The WLDW line segregated for three MHC haplotypes, B15, B19, and B21, and for the sex-linked dwarf gene, DW. Chicks were challenged at 4 wk of age with a high dose of Eimeria tenella (150,000 oocysts) and slaughtered 8 d postinoculation. Innate resistance was assessed individually by measures of lesion score, mortality, and body weight gain at slaughter, and plasma coloration 4 d postinoculation. Large differences in resistance to E. tenella were observed between lines. The Fayoumi line appeared clearly as the most resistant line, showing no mortality, less severe lesions than other lines, and a 30% reduction of growth as compared to control birds. The WLDW line was the most susceptible, with 27% mortality and a 85% reduction in growth. No major effect of MHC or dwarfism on resistance to E. tenella was found.

Eimeria tenella: cloning and characterization of cDNA encoding a S3a ribosomal protein.

A lambda Zap II cDNA library was constructed from Eimeria tenella first- generation schizonts mRNA and screened with a mouse serum raised against this parasitic stage. This serum identified a clone encoding a S3a ribosomal protein (EtS3a). The 858-bp cDNA fragment, containing the entire parasitic gene encoded a highly basic protein of 264 amino acids (aa) with a molecular weight of 29.780kDa. Based upon amino acid sequence comparison, EtS3a is highly homologous to v-fos transformation effector (encoded by the fte-1 gene) and cyc-07 (a plant homologue of fte-1) and similar to the yeast MFT1 (encoded by the mitochondrial fusion targeting gene). The expressions of mammalian fte-1, plant cyc-07 and yeast MFT1 have all been shown to be cell-cycle-regulated and involved in protein synthesis at the level of the ribosome. Since EtS3a expression is also developmentally regulated, we suggest that this gene product is a functional homologue of fte-1, cyc-07 and MFT1 and an important molecule regulating the development of Eimeria tenella.

Immune responses and protective effect in mice vaccinated orally with surface sporozoite protein of Eimeria falciformis in ISCOMs.

Immunostimulating complexes (ISCOMs) were built after treatment of a purified surface protein from Eimeria falciformis sporozoites with a palmitic acid derivation, leading to a high ratio (33-64%) of P27 incorporation in these cage-like structures. P27 kept its antigenicity after incorporation in ISCOMs, which induced, after iterative intubations by the oral route to groups of mice, a systemic IgG response, a local IgA response, and a local enhanced cellular response as demonstrated by lymphoproliferation of mesenteric lymph node cells upon in vitro stimulation with antigen. This immunization (120 micrograms in six oral doses at 2-day intervals) afforded mice a partial protection (60%) against a subsequent 400 oocyst challenge. The reduction in daily oocyst excretion was corroborated by significantly different weight losses between immunized and control mice on days 9 and 10 postinfection and the subsequent death of these control mice. These observations provide the first application of ISCOMs to parasitic intestinal diseases.

Production and characterization of monoclonal antibodies directed against Eimeria falciformis and cross-reactive with sporozoites from two species of avian coccidia.

Monoclonal antibodies (mAbs) were obtained against the surface antigens of the Eimeria falciformis sporozoite by immunizing mice with whole homogenized sporozoite. The hybridomas were selected by their reactivities against oocyst extracts, then against glutaraldehyde-treated sporozoites. Three mAbs recognized both the surface of E. falciformis, E. tenella, and E. acervulina and their refractile bodies, whereas a fourth mAb recognized only one epitope on the refractile bodies. All mAbs bound to the same immunoaffinity-purified antigens in Western-blot analysis (P27 for E. falciformis and P25 for E. tenella and E. acervulina). Thus, the mAbs define at least two shared epitopes between sporozoite antigens from different eimerian species. Two of these mAbs are involved in the in vitro phagocytosis of E. falciformis sporozoites by macrophages and also in their lysis by neutrophils. Altogether, these properties showed that the four mAbs came from different activated B-cells. The P27 antigen recognized by our mAbs represents a major target of the in vitro destructive immune response.

The immunodominant Eimeria acervulina sporozoite antigen previously described as p160/p240 is a 19-kilodalton antigen present in several Eimeria species.

A lambda Zap II cDNA expression library, constructed from Eimeria acervulina (PAPa46 strain) sporulated oocyst stage, was screened with sera raised to E. acervulina or Eimeria tenella oocysts in order to isolate clones coding for antigens common to the two species. Most of the clones isolated were derived from the same gene. Antisera raised to a recombinant glutathione-S-transferase fusion protein 1P reacted with an antigen of 19 kDa in immunoblot of E. acervulina sporulated and unsporulated oocysts. Immunofluorescence of E. acervulina sporozoites indicated that the antigen is located in the cytoplasm. The anti-1P antisera reacted on immunoblots of E. tenella with a 19-kDa antigen and by immunofluorescence on E. tenella, Eimeria maxima and Eimeria falciformis sporozoites, indicating that the antigen is conserved in Eimeria species. DNA sequencing indicated that the sequence was almost identical to that of clone cSZ1 previously described by Jenkins et al. using E. acervulina strain #12. The 1P insert hybridized to a 1150-nt mRNA from E. acervulina PAPa46 strain and strain #12, a size consistent with the observed molecular weight of the protein.

Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

Protective oral immunization of chickens against Eimeria tenella with sporozoite surface antigens.

Antigens were extracted from the surface of Eimeria tenella sporozoites with a solution containing Triton X 100 (1%), sodium dodecyl sulphate (0.5%) Na deoxycholate (1%) and EDTA (1 mM). After removal of the detergents, these surface antigen preparations conferred an immunity that protected chickens against a subsequent infection (10(4) sporulated oocysts). The best results were obtained after two 250 micrograms injections of Al(OH)3 adsorbed antigens (oocyst output per g caecal material on Day 7 post infection: 2.39 x 10(7) +/- 0.32 x 10(7) oocysts for controls and 7.37 +/- 10(6) +/- 3.19 x 10(6) oocysts for vaccinated birds) and after four gastric intubations of liposome entrapped antigens (oocysts output on Day 7 postinfection: 2.75 x 10(6) +/- 2.02 x 10(6) g-1 caecal material). These results represented respectively 70 and 88% protection indexes. Studies on the systemic and local antibody response after one or several infections of chickens with the parasite indicated at least 20 different molecules in the detergent antigens which are classified after immunoblotting according to their properties.

[Anticoccidial vaccines. Review and perspectives]. / Vaccins anticoccidiens. Bilan et perspectives.

Coccidioses are a major problem for the poultry industry. Currently, the diseases are controlled by drug chemoprevention, but the development of chemoresistant strains has for several years incited investigators to search for a vaccine. Several methods were investigated: oocyst administration at low and controlled doses, administration of strains which had been attenuated by physical or biochemical means, immunization with extracted or recombinant antigens. For some years to come genetically selected precocious live strains of Eimeria which are now commercially available will be useful as vaccines; but in the future, genetically engineered antigens will provide new opportunities for a successful vaccination.

[Eimeria tenella and Eimeria acervulina: an antigenic stimulation in vitro of cells from immune chickens induced by adoptive transfer of protection against avian coccidiosis]. / Eimeria tenella et Eimeria acervulina: une stimulation antigénique in vitro des cellules provenant de poulets immuns induit le transfert adoptif de la protection contre les coccidioses aviaires.

The transfer of 5 x 10(7) or 10(8) spleen cells from E tenella-infected chickens to virgin animals after 12-20-h in vitro stimulation with whole sporozoite homogenates confers significant protection to recipients. The oocyst contents of ceca on d 7 post-infection with 20,000 E tenella oocysts were (1.33 +/- 1.10) x 10(6) in chickens which received 5 x 10(7) immune cells after 20-h in vitro stimulation and (4.64 +/- 2.85) x 10(6) in chickens receiving 5 x 10(7) stimulated cells from normal chickens (85% protection). Adoptive transfer by spleen cells revealed an asymmetric cross-protection between E tenella and E acervulina. Spleen cells from E tenella immune chickens protected only against a subsequent infection with the same parasite, while spleen cells from E acervulina immune chickens protected against infection with E acervulina (78%) but also against infection with E tenella (68% protection). The common antigen permits better stimulation, but common surface sporozoite antigens purified from E tenella sporozoites via anti-E acervulina biliary antibodies are capable of stimulating both types of cells without, however, changing their properties.

In vitro interactions between murine neutrophils and Eimeria falciformis sporozoites.

The in vitro interactions between elicited mouse peritoneal neutrophils, antibodies and newly excysted sporozoites of Eimeria falciformis resulted in lysis of the parasite. This lysis required the presence of a heat-labile component of normal mouse serum, and was antibody- and cell-concentration-dependent. Under optimal conditions (serum dilution = 1/192, effector cell/sporozoite = 10/1) this lysis, which began after incubation at 37 degrees C for 4 h, was nearly complete after 18 h. It began by opsonization of the sporozoites by antibodies and complement. Inhibition studies performed with inhibitors of neutrophil function did not enable us to determine the mechanism of this extracellular lysis (oxidative respiratory burst or enzyme release), since only metal chelators, lysosomotropic reagents and compounds known to interfere with adenylate cyclase activity were truly inhibitory.

In vitro interactions between murine macrophages and Eimeria falciformis sporozoites.

A short-term (2 h) assay was used to investigate the in vitro fate of Eimeria falciformis sporozoites in murine peritoneal macrophages. In minimal medium, uptake of sporozoites was low by both normal (naive) and immune macrophages. However, when heat-inactivated serum from immune mice was added to the incubation medium, sporozoite uptake was much more efficient. Sporozoite lysis was observed only in immune macrophages and required both antibodies and complement. Pretreatment of immune macrophages with chloroquine inhibited sporozoite lysis and resulted in an accumulation of sporozoites within the cells. Immunoabsorption assays revealed that IgG2a was the major isotype mediating entry of sporozoites into macrophages, both in early (6 days post-primary) and late (second) infections.