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Background: The phylum Apicomplexa comprises a wide variety of parasites of significant medical and economic relevance. These parasites have extremely different host and tissue tropisms; for example Toxoplasma gondii can invade virtually any nucleated cell and infect almost all warm-blooded vertebrates, whereas Eimeria tenella infects only chickens and is restricted in its growth to epithelial cells of the caecum. Proteins released from the microneme secretory organelles (MICs) are critical for apicomplexan invasion of host cells and allow parasites to bind a diverse range of host cell oligosaccharide epitopes. MICs bear modular arrangements of sequences with adhesive proteins and interestingly the sialic-acid binding MAR (microneme adhesive repeat) domain containing proteins (MCPs) are suggested to make significant contributions to the different host and tissue tropisms of T. gondii and E. tenella. Results: In this study, we evaluated the binding capacity of Type I MAR domains from novel E. tenella MCPs. Variants of the previously described HxT motif were analysed showing that HxT and VxT variants bind, whereas HxS and YxE variants did not. One of these MCP containing a single MAR (EtMCP2) showed an apical localization when expressed as a fusion with the fluorescent reporter mCherry in transgenic populations and a similar pattern of transcripts per zoite during endogenous development in vitro as the well-characterised microneme protein EtMIC2. Conclusions: Variation in the binding properties of the MAR of different EtMCPs was confirmed and their ability to bind a wider range of sialic acids and terminal linkages should be studied. In addition, transgenesis technology has been used for first time in Eimeria parasites as a rapid tool for the study of endogenous protein localization by fusion with a fluorescent reporter. [ABSTRACT FROM AUTHOR]

Background: Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens. Results: In this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5'Et-Actin or 5'Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5'Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens. Conclusions: E. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector. [ABSTRACT FROM AUTHOR]

Background: Chicken is fast becoming the world's most consumed meat. As a consequence poultry health is more important now than ever before, with pathogens of chickens recognised as serious threats to food security. One such threat are Eimeria species parasites, protozoa which can cause the disease coccidiosis. Eimeria can compromise economic poultry production and chicken welfare, and have serious consequences for poor livestock keepers. Seven Eimeria species that infect chickens are recognised with a global enzootic distribution. More recently three cryptic Operational Taxonomic Units (OTUx, y and z) have been described in populations of Eimeria recovered from chickens in Australia. Two of the three OTUs have also been detected in sub-Saharan Africa, but their occurrence, pathology and the risk they pose is largely unknown. Results: Nigeria has witnessed a dramatic expansion in poultry production and is now the largest poultry producer in Africa. Here, faecal samples collected from nine of 12 commercial chicken farms sampled in Kaduna state, Nigeria, were found to contain eimerian oocysts. After amplification by in vivo propagation all three cryptic OTU genotypes were detected using polymerase chain reaction (PCR), including OTUy for the first time outside of Australia. Comparison with a widely used, established Eimeria species-specific PCR assay revealed failure to detect the OTU genotypes. Conclusions: All three of the Eimeria OTU genotypes appear to be common in north-western Nigeria. The failure of a leading species-specific molecular assay to detect these genotypes indicates a risk of false negative Eimeria diagnosis when using molecular tools and suggests that the spatial occurrence of each OTU may be far wider than has been recognised. The risk posed by these novel genotypes is unknown, but it is clear that a better understanding of Eimeria occurrence is required together with the validation of effective diagnostics. [ABSTRACT FROM AUTHOR]

Background: Proteases regulate pathogenesis in apicomplexan parasites but investigations of proteases have been largely confined to the asexual stages of Plasmodium falciparum and Toxoplasma gondii. Thus, little is known about proteases in other Apicomplexa, particularly in the sexual stages. We screened the Eimeria tenella genome database for proteases, classified these into families and determined their stage specific expression. Results: Over forty protease genes were identified in the E. tenella genome. These were distributed across aspartic (three genes), cysteine (sixteen), metallo (fourteen) and serine (twelve) proteases. Expression of at least fifteen protease genes was upregulated in merozoites including homologs of genes known to be important in host cell invasion, remodelling and egress in P. falciparum and/or T. gondii. Thirteen protease genes were specifically expressed or upregulated in gametocytes; five of these were in two families of serine proteases (S1 and S8) that are over-represented in the coccidian parasites, E. tenella and T. gondii, distinctive within the Apicomplexa because of their hard-walled oocysts. Serine protease inhibitors prevented processing of EtGAM56, a protein from E. tenella gametocytes that gives rise to tyrosine-rich peptides that are incorporated into the oocyst wall. Conclusion: Eimeria tenella possesses a large number of protease genes. Expression of many of these genes is upregulated in asexual stages. However, expression of almost one-third of protease genes is upregulated in, or confined to gametocytes; some of these appear to be unique to the Coccidia and may play key roles in the formation of the oocyst wall, a defining feature of this group of parasites. [ABSTRACT FROM AUTHOR]

Background: Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis., Results: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis., Conclusions: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.