Eimeria tenella (Eucoccidiorida): a quantitative assay for sporozoite infectivity in vivo

A quantitative technique for the assessment of sporozoite infectivity in vivo, using intra-cecal inoculation of Eimeria tenella sporozoites, has been developed. Evaluation of the infection using cecal lesion scores and oocyst counts showed that this technique should be useful for the quantitation of sporozoite viability and thus for the anti-sporozoite activity of different treatments prior to inoculation. Pre-treatment of sporozoites with heat-inactivated hyperimmune antisera neutralized sporozoite infectivity in vivo and indicated that antibodies in the absence of complement inhibited sporozoite infectivity in vivo.     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Towards a reference map of Eimeria tenella sporozoite proteins by two-dimensional electrophoresis and mass spectrometry

Eimeria tenella is a parasite of great importance as a disease causing agent in the poultry industry. Until recently, biological studies have focused on specific proteins, some of which play an important role in the parasite life cycle. Post-genomic studies will make it possible to understand the complexity of the parasites and their interactions with host cells. Here we present a systematic reference map of the proteins from E. tenella sporozoites. The proteins expressed at the sporozoite stage were resolved between isoelectric points 3-10 and 4-7. They were systematically identified using mass spectrometry and 16 known Eimeria sporozoite proteins were identified on two-dimensional maps. Peptide fragmentation data from mass spectrometry were compared to single and consensus expression sequence tags in databases and to the E. tenella genome (not annotated). Among the set of unknown proteins analysed, 12 new assignments were proposed on the basis of similarities with Apicomplexa proteins. In order to define sporozoite proteins as potential targets for coccidiosis therapy, proteins were studied according to their relative abundance and immunogenicity in the sporozoite. Immunoblots of sporozoite 2D maps with chicken sera were performed and approximately 50 proteins were defined as antigens. It was shown that abundance and immunogenicity are not related in the sporozoite stage. Perspectives of gene prediction and completion of the genome annotation by a proteomic approach is discussed.     

Production of monoclonal antibodies specific for Eimeria tenella microgametocytes

A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.     

Eimeria tenella: identification of secretory and surface proteins from expressed sequence tags

To identify new vaccine candidates, Eimeria tenella expressed sequence tags (ESTs) from public databases were analysed for secretory molecules with an especially developed automated in silico strategy termed DNAsignalP. A total of 12,187 ESTs were clustered into 2881 contigs followed by a blastx search, which resulted in a significant number of E. tenella contigs with homologies to entries in public databases. Amino acid sequences of appropriate homologous proteins were analysed for the occurrence of an N-terminal signal sequence using the algorithm signalP. The resulting list of 84 entries comprised 51 contigs whose deduced proteins showed homologies to proteins of apicomplexan parasites. Based on function or localisation, we selected candidate proteins classified as (i) secreted proteins of Apicomplexa parasites, (ii) secreted enzymes, and (iii) transport and signalling proteins. To verify our strategy experimentally, we used a functional complementation system in yeast. For five selected candidate proteins we found that these were indeed secreted. Our approach thus represents an efficient method to identify secretory and surface proteins out of EST databases.     

Eimeria tenella: Local Antibodies and Interactions with the Sporozoite Surface

.Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10³ oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10³ oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and imtnunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results. The immunofluorescence studies reveal the extremely labile nature of the sporozoite surface antigens and support the hypothesis that naturally elaborated IgA antibodies are involved in the modulation of complexed sporozoite surface antigens.     

Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis

Hybridoma cell lines, which secreted antibodies directed against two different strains of Eimeria tenella and one strain of E. mitis, were produced by fusion of spleen cells from sporozoite-immunized Balb/cByJ mice with P3-X63-Ag8 myeloma cells. The antibodies demonstrated at least eight different binding patterns on or in air-dried sporozoites as determined by the indirect immunofluorescent antibody (IFA) test. These patterns varied from a general internal fluorescence similar to that seen with sporozoites exposed to hyperimmune chicken serum, to fluorescence observed on the tip, pellicle, and refractile body of the parasite. Five cloned, antibody-secreting cell lines were successfully established. Four of these clones produced antibody that reacted only with various strains of E. tenella and cross-reacted with no other species of coccidia. The fifth clone produced antibody directed against only E. mitis and did not react with any other coccidial species.     

Establishment of Eimeria tenella (local isolate) in chicken embryos

Development of an in vitro Eimeria (E.) tenella model could be valuable as a tool for vaccine, coccidiostats or molecular biology research. 1.0 × 10,000 sporozoites per 0.1 mL were inoculated into the allantoic cavity of ten-day-old chicken embryos. The complete life-cycle of E. tenella was accomplished in eight-nine days at 37 °C and 70% humidity. The addition of 100 U insulin to the embryos could remarkably improve the output of oocysts. The development of the parasite within the embryos was systematically observed, allowing guidelines to be set regarding the appropriate times at which different developmental stages of the parasite may be sampled.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and 125I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.     

Eimeria tenella: utilization of a nasal vaccine with sporozoite antigens incorporated into Iscom as protection for broiler breeders against a homologous challenge

The aim of this study was to evaluate a nasal vaccine using antigens derived from sporozoites of Eimeria tenella incorporated into Iscom to protect broiler chicks. Forty-five one-day-old chickens (Cobb), unvaccinated against coccidiosis, were used in this experiment. The birds were maintained in separated battery cages and divided into three groups: G1 (n = 15), G2 (n = 15), and G3 (n = 15). G1 received 50 μg of sporozoites + Iscom vaccine, G2 received Iscom without antigens, and G3 received only PBS. The treatments were administered by nasal route on days 0, 7, and 21 of the experiment. On the 28th day, all birds were challenged with 105 sporulated oocysts of E. tenella. On the challenge day, three birds from each group were euthanized to evaluate lymphocyte proliferation. Lesion scores were obtained from five birds from each group, 7 days after challenge. The remaining animals were euthanized on the 50th day. The mean lymphocyte proliferation responses were significantly different (P = 0.03); G1 was 2.3-2.6 times more elevated than G2, and G3 (P < 0.001). 83% of the birds from G1 showed an IgY antibody reaction by ELISA at challenge. The means for oocysts shedding were 16,890 ± 20,511, 48,080 ± 50,047, and 65,020 ± 74,461, for G1, G2 and G3 birds, respectively. There was no significant difference (P > 0.17) in oocysts shedding between groups. However, the G1 and G2 chicks demonstrated reduction in percentage of oocyst shedding when compared to control birds (G3) by 74.02% and 26.05%, respectively. The average lesion scores were G1 = 0.4, G2 = 1, and G3 = 2. This study demonstrated that the lowest lesion score and oocyst shedding were observed in the birds from the group that received antigens derived from sporozoite with an Iscom adjuvant (G1). These results suggest that this vaccine can induce protection against avian coccidiosis.     

Production and partial characterization of monoclonal antibodies specific for the gamonts of Eimeria tenella.

Thirteen hybridomas secreting monoclonal antibodies (Mabs) specific for the sexual stages (gamonts) of Eimeria tenella were produced by fusing spleen cells of gamont-immunized RBF/Dn mice with FOX-NY myeloma cells. A Mab subisotype profile revealed 1 IgG2a and 12 IgG1. All Mabs demonstrated a similar binding pattern when incubated with parasitic gamonts as determined by the indirect fluorescent antibody test. Ascitic fluid containing Mab (GD9 (IgG1) was produced and used to immunize chicks passively per os. There was a 34% decrease (P less than 0.05) in oocyst output from immunized chicks when compared to control chicks. Passively immunized chicks also had reduced cecal lesion scores when compared to control chicks. These results suggest that Mab GD9 partially inhibited the fertilization process of E. tenella.     

Eimeria tenella: Host Specificity in Gallinaceous Birds

SYNOPSIS. Eight species representing 8 genera of gallinaceous birds were used: Alectoris graeca; Colinus virginianus; Coturnix coturnix; Gallus gallus; Meleagris gallopavo; Numidia meleagris; Pavo cristatus; Phasianus colchicus. Three-week-old birds were dosed with sporulated oocysts of Eimeria tenella Beltsville strain. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 infected birds and uninoculated controls of each species were killed by cardiac exsanguination. Pieces of intestines were fixed and examined for stages of E. tenella as stained paraffin sections or indirect fluorescent antibody preparations. Oocyst counts were made in droppings collected for the first 6 days of the patent period. Sporozoites were found in the lamina propria of some birds of 5 species at 4 hr postinoculation, but no stages were found thereafter except in the breeds of G. gallus and A. graeca. At 144 and 168 hr postinoculation, a few macrogametes were found in the ceca of 2 A. graeca , but no oocysts were found in the feces. No statistical difference was found between the number of oocysts produced/bird in the breeds of G. gallus examined. It is evident from these observations that E. tenella did not complete its life cycle in several close phylogenetic relatives of G. gallus , even though in other studies this parasite was found to complete its life cycle in cell cultures derived from the same birds.     

A genetic linkage map for the apicomplexan protozoan parasite Eimeria maxima and comparison with Eimeria tenella

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of ∼14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10 years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines.