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Four complete mitochondrial (mt) sequences from a single-oocyst-derived line of Eimeria mitis USDA 50 were obtained (three from cloned whole-genome PCR products, one from directly sequenced whole-genome PCR product). The mt genome is 6,408 bp long with three genes (CytB, cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit III (COIII)) and many rDNA fragments (large subunit rDNA 13, small subunit rDNA 10); organisation was identical to other Eimeria sp. mt genomes. Conserved start codon positions for both COI and COIII are suggested for all Eimeria mt genomes; these start codon positions exist and may also be conserved, in related apicomplexan parasites. Within the three separate cloned PCR products of near-complete mt genomes, there were 26 nucleotide differences (collectively) compared to the directly sequenced mt genome. These changes appear to be base misincorporations during PCR. Direct sequencing of long PCR amplification products may be more likely to generate accurate mt genomic sequences than cloning and subsequent sequencing.

The near complete mitochondrial (mt)genome for Cyclospora cayetanensis is 6184 bp in length with three protein-coding genes (Cox1, Cox3, CytB) and numerous lsrDNA and ssrDNA fragments. Gene arrangements were conserved with other coccidia in the Eimeriidae, but the C. cayetanensis mt genome is not circular-mapping. Terminal transferase tailing and nested PCR completed the 5’-terminus of the genome starting with a 21bp A/T-only region that forms a potential stem-loop. Regions homologous to the C. cayetanensis mt genome 5’-terminus are found in all eimeriid mt genomes available and suggest this may be the ancestral start of eimeriid mt genomes.

BackgroundClinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo).MethodsSix single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome.ResultsComplete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens.ConclusionsMitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology.

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