Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome—which appears to be representative of the genome—is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.

BackgroundIn Malaysia, the domestic water buffaloes (Bubalus bubalis) are classified into the swamp and the murrah buffaloes. Identification of these buffaloes is usually made via their phenotypic appearances. This study characterizes the subspecies of water buffaloes using karyotype, molecular and phylogenetic analyses. Blood of 105 buffaloes, phenotypically identified as swamp, murrah and crossbred buffaloes were cultured, terminated and harvested using conventional karyotype protocol to determine the number of chromosomes. Then, the D-loop of mitochondrial DNA of 10 swamp, 6 crossbred and 4 murrah buffaloes which were identified earlier by karyotyping were used to construct a phylogenetic tree was constructed.ResultsKaryotypic analysis confirmed that all 93 animals phenotypically identified as swamp buffaloes with 48 chromosomes, all 7 as crossbreds with 49 chromosomes, and all 5 as murrah buffaloes with 50 chromosomes. The D-loop of mitochondrial DNA analysis showed that 10 haplotypes were observed with haplotype diversity of 0.8000 ± 0.089. Sequence characterization revealed 72 variables sites in which 67 were parsimony informative sites with sequence diversity of 0.01906. The swamp and murrah buffaloes clearly formed 2 different clades in the phylogenetic tree, indicating clear maternal divergence from each other. The crossbreds were grouped within the swamp buffalo clade, indicating the dominant maternal swamp buffalo gene in the crossbreds.ConclusionThus, the karyotyping could be used to differentiate the water buffaloes while genotypic analysis could be used to characterize the water buffaloes and their crossbreds.

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.

Coccidiosis, caused by the Eimeria species, greatly affects the poultry industry. Severity of the disease varies depending on the identity of the infecting parasites, encouraging identification of Eimeria species circulating on a farm as a valuable component of chicken management. Conventional methods of Eimeria species identification are time consuming and can be subjective in nature. Given these limitations, molecular approaches have been developed for specific detection of Eimeria species. In this study, faecal samples were collected from commercial broiler farms and subjected to microscopic examination for Eimeria occurrence. Eimeria species were putatively identified by morphological characterisation and grouped into three categories based on oocyst size. Molecular detection of Eimeria species occurrence in these samples was then performed using two published PCR assays (the individual components of a SCAR-based multiplex PCR, and assays developed for quantitative PCR, termed PCR-SCAR 1 and PCR-SCAR 2 here) and a LAMP assay. Comparison of the results obtained demonstrated that the three molecular methods were capable of detecting all Eimeria species of the reference Houghton strain, but showed varying efficiencies in detecting Malaysian field isolates. PCR-SCAR 2 was found to be the most effective, detecting all seven Eimeria species and indicating the presence of Eimeria parasites in most flocks. Differences in the ability of the molecular methods to detect Eimeria may be a consequence of sequence divergence between isolates from different regions, implying that development of region-specific methods using local Eimeria strains may be required to improve the efficiency of molecular assays for Eimeria detection.