Phylogenies based on combined mitochondrial and nuclear sequences conflict with morphologically defined genera in the eimeriid coccidia (Apicomplexa).

Partial mitochondrial (mt) cytochrome c oxidase subunit I (COI) and near-complete nuclear (nu) 18S rDNA sequences were obtained from various eimeriid coccidia infecting vertebrates. New and published sequences were used in phylogenetic reconstructions based on nu 18S rDNA, mt COI and concatenated sequence datasets. Bayesian analyses of nu 18S rDNA sequences used secondary structure-based alignments with a doublet nucleotide substitution model; the codon nucleotide substitution model was applied to COI sequences. Although alignment of the mt COI sequences was unambiguous, substitution saturation was evident for comparisons of COI sequences between ingroup (eimeriid) and outgroup (sarcocystid) taxa. Consequently, a combined dataset applying partition-specific analytical and alignment improvements was used to generate a robust molecular phylogeny. Most eimeriid parasites that infect closely related definitive hosts were found in close proximity on the resulting tree, frequently in a single clade. Whether this represents coevolution or co-accommodation or a combination remains an open point. Unlike host associations, basic oocyst configuration (number of sporocysts per oocyst and sporozoites per sporocyst) was not correlated with phylogeny. Neither 'Eimeria-type' nor 'Isospora-type' oocyst morphotypes formed monophyletic groups. In the combined dataset tree (representing only a tiny fraction of described eimeriid coccidia), at least 10 clades of Eimeria spp. would need to be re-assigned to nine distinct genera to resolve their paraphyly. The apparent lack of congruence between morphotype and genotype will require taxonomists to balance nomenclatural stability and diagnostic ease against the ideal of monophyletic genera. For now, recognition of paraphyletic eimeriid genera defined by basic oocyst configuration may be necessary for reasons of taxonomic stability and diagnostic utility. Future taxonomic revisions to produce monophyletic eimeriid genera will ultimately require the identification of reliable phenotypic characters that agree with the molecular phylogeny of these parasites or, less optimally, acceptance that genotyping may be needed to support monophyletic supraspecific taxonomic groups.

Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys.

BACKGROUND: Clinical 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). METHODS: Six 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. RESULTS: Complete 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. CONCLUSIONS: Mitochondrial 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.

Description of two new Isospora species causing visceral coccidiosis in captive superb glossy starlings, Lamprotornis superbus (Aves: Sturnidae).

Isospora greineri sp. n. and Isospora superbusi sp. n. are described from captive superb glossy starlings, Lamprotornis superbus, from the Toronto Zoo succumbing to visceral coccidiosis. Sequence data from nuclear 18S recombinant DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (COI) loci from sporulated oocysts and infected tissues (liver, lung, or spleen) demonstrated two distinct Isospora sp. genotypes that varied in their relative abundance. In the tissues of one affected bird, as well as its associated fecal sample, two distinct COI sequences (1.7% divergence) and two distinct 18S rDNA sequences (0.6% divergence) were found at almost the same abundance; in other specimens, one of the 18S and one of the COI sequences were less abundant than the other. In the tissues of some birds, only a single COI and single 18S sequence were present. In all cases, the same pair of 18S rDNA and COI sequences fluctuated in abundance in parallel, indicating that there were two distinct species present rather than one species with more than one COI or 18S locus. The oocysts of these new species cannot be differentiated morphologically. Sporulated oocysts of both were spherical to subspherical measuring 17.7 ± 0.22 µm by 17.1 ± 0.20 µm with a mean L/W ratio of 1.03 ± 0.004. Sporocysts were ovoid measuring 13.5 ± 0.17 µm by 9.3 ± 0.15 µm with a mean L/W ratio of 1.4 ± 0.02. Sporocysts had a small Stieda body with indistinct sub-Stieda body; each sporocyst had a compact residuum. Two morphologically similar but genetically divergent Isospora species were shown to cause simultaneous enteric and extraintestinal infections in captive superb glossy starlings.

Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks.