Phylogenetic relationships among rodent Eimeria species determined by plastid ORF470 and nuclear 18S rDNA sequences

Phylogenetic analyses for 10 rodent Eimeria species from different host genera based on plastid ORF470 and nuclear 18S rDNA sequences were done to infer the evolutionary relationships of these rodent Eimeria species and their correlation to morphology and host specificity. The phylogenies based on both data sets clearly grouped the 10 rodent Eimeria species into two major lineages, which reflect more their morphological differences than host specificity. Species in lineage A have spheroidal to subspheroidal sporulated oocysts, are similar in size (18-29 x 17-23; xbar = 22 x 20 microm), have an oocyst residuum and one-two polar granules; these include Eimeria albigulae (Neotoma), Eimeria arizonensis (Peromyscus, Reithrodontomys), Eimeria onychomysis (Onychomys) and Eimeria reedi (Perognathus). Species in lineage B, including Eimeria falciformis (Mus), Eimeria langebarteli (Reithrodontomys), Eimeria nieschulzi (Rattus), Eimeria papillata (Mus), Eimeria separata (Rattus) and Eimeria sevilletensis (Onychomys) have different shapes (ovoid, ellipsoid, elongated ellipsoid, etc.), differ greatly in size (10-27 x 9-24; xbar = 19 x 16 microm) and all lack an oocyst residuum. Thus, The oocyst residuum was the most determinant feature that differentiated the two lineages. The accession numbers of ORF470 of E. albigulae, E. arizonensis, E. falciformis, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis, E. langebarteli are AF311630-AF311639 and 18S rDNA of E. langebarteli, E. papillata, E. reedi, E. separata, E. sevilletensis are AF311640-AF311644.     

Eimeria species (Apicomplexa: Eimeriidae) infecting Eliomys quercinus in an Alpine habitat

Coccidian parasites were detected in an Alpine population of the garden dormouse (Eliomys quercinus), and 55-82% of the fecal samples collected during a two-year study (2000 and 2002) contained one or two eimerian species. We report the presence of Eimeria myoxi and confirm for the first time the presence of Eimeria melanuri in the garden dormouse. These Eimeria species can be considered common parasites of the garden dormouse and the Asian garden dormouse. The high prevalence might be due to group hibernation by the dormice.     

Molecular characterization of Eimeria species in macropods

A total of 597 faecal samples were collected from western grey kangaroos (Macropus fuliginosus), Euros (M. robustus), red kangaroos (M. rufus) in Western Australia and Eastern Grey Kangaroos (M. giganteus) from Victoria and screened for the presence of Eimeria by PCR at the 18S ribosomal RNA (rRNA) locus. The overall prevalence was 24.3% (145/597). At the 18S rRNA locus, sequences were obtained for 25 of the 145 positives. Phylogenetic analysis indicated that all the macropod-derived Eimeria species grouped in a separate marsupial clade that included Eimeria trichosuri from brushtail possums. At least 6 different clades were identified within the marsupial isolates and many of the genotypes identified are likely to be valid species, however morphological and biological data need to be collected to match sequences to previously characterized Eimeria species or identify if they are new species.     

Eimeria species (Apicomplexa: Eimeriidae) from arctic ground squirrels (Spermophilus parryii) and red squirrels (Tamiasciurus hudsonicus) in Alaska and in Siberia, Russia

Fecal samples from arctic ground squirrels (Spermophilus parryii) collected in Alaska (n = 90) and Russia (n = 46) and from red squirrels (Tamiasciurus hudsonicus) in Alaska (n = 35) were examined for the presence of Eimeria spp. (Apicomplexa: Eimeriidae). Four species were recovered from arctic ground squirrels, including Eimeria callospermophili (prevalence = 18%), Eimeria cynomysis (23.5%), Eimeria lateralis (19%), and Eimeria morainensis (77%). A single species, Eimeria tamiasciuri (91%), was recovered from red squirrels. Eimerians recovered from arctic ground squirrels represent new host records, and the single species from red squirrels is a new geographic record. Alaskan arctic ground squirrel prevalence was higher for E. callospermophili (Alaska = 22% vs. Russia = 9%), E. cynomysis (34% vs. 2%), and E. lateralis (27% vs. 4%), but not E. morainensis (78% vs. 76%).     

Phylogenetic relationships among Eimeria spp. (Apicomplexa, Eimeriidae) infecting rabbits: evolutionary significance of biological and morphological features

Monophyly of all 11 valid Eimeria species from rabbits (Oryctolagus cuniculus Linnaeus, 1758) was revealed based on nuclear 18S rDNA sequence data. This finding implies that these species, which vary considerably in terms of their morphology and biology, diversified on a single host or several closely related species. Phylogenetic analysis divided rabbit Eimeria species into 2 sister lineages, corresponding to the presence/absence of the oocyst residuum. Other morphological or biological traits (oocyst shape and size, presence/absence of oocyst inner structures, pathogenicity, infection site, pre-patent and patent periods, sporulation time, and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.     

Phylogenetic position of Eimeria antrozoi, a bat coccidium (Apicomplexa: Eimeriidae) and its relationship to morphologically similar Eimeria spp. from bats and rodents based on nuclear 18S and plastid 23S rDNA sequences.

Partial plastid 23S and nuclear 18S rDNA genes were amplified and sequenced from 2 morphologically similar Eimeria species. E. antrozoi from a bat (Antrozous pallidus) and E. arizonensis from deer mice (Peromyscus spp.), as well as some other Eimeria species from bats and rodents. The phylogenetic trees clearly separated E. antrozoi from E. arizonensis. The phylogenies based on plastid 23S rDNA data and combined data of both plastid and nuclear genes grouped 2 bat Eimeria and 3 morphologically similar Eimeria species from rodents into 2 separate clades with high bootstrap support (100%, 3 rodent Eimeria species; 72-97%, 2 bat Eimeria species), which supports E. antrozoi as a valid species. The rodent Eimeria species did not form a monophyletic group. The 2 bat Eimeria species formed a clade with the 3 morphologically similar rodent Eimeria species (E. arizonensis, E. albigulae, E. onychomysis, all from cricetid rodents) with 100% bootstrap support, whereas 2 other rodent Eimeria species (E. nieschulzi, E. falciformis, from murid rodents) formed a separate clade with 100% bootstrap support. This suggests that the 2 Eimeria species from bats might be derived from rodent Eimeria species and may have arisen as a result of lateral host transfer between rodent and bat hosts.     

A new eimeriid (Apicomplexa) species from endangered Attwater's prairie chickens (Tympanuchus cupido attwateri) in Texas

The Attwater's prairie chicken (APC; Tympanuchus cupido attwateri Bendire, 1894) has been a federally listed endangered species since 1967. Several captive propagation programs consisting of small populations are being used to keep this species from extinction. Fecal samples were collected from APCs in April 2007 and again in August 2008 from 2 separate captive propagation facilities in Texas after clinical signs of coccidiosis were observed. One Eimeria species was observed (Eimeria attwateri), which we describe as a putative new species. Sporulated oocysts are ellipsoidal, 30.0 × 18.4 (27.4-31.3 × 16.0-22.4) μm. Oocysts have a smooth wall 0.7 μm thick and lack both a micropyle and oocyst residuum, but 1 ellipsoidal polar granule is present, 2.3 × 1.9 (2.1-2.4 × 1.7-2.0) μm. Sporocysts have a nipple-like Stieda body with a rounded opposite end and are 14.0 × 7.1 (10.2-16.8 × 6.0-9.2) μm. The sporocysts contain a sporocyst residuum usually consisting of 2-4 dispersed globules, and each sporozoite contains 2 large posterior spheroid refractile bodies 3.4 μm wide. Nucleotide sequence amplified from the 18S rDNA does not match any DNA sequence information for publicly available Eimeria species, and phylogenetic reconstructions place this species with other eimerians from Galliformes. The discovery of a potentially pathogenic species of Eimeria in captive APCs is of great importance, and managers should be aware of the potential devastating effect(s) this parasite could have on the APC conservation programs.     

Host-Parasite Incongruences in Rodent Eimeria Suggest Significant Role of Adaptation Rather than Cophylogeny in Maintenance of Host Specificity

The degree of host specificity, its phylogenetic conservativeness and origin are virtually unknown in Eimeria. This situation is largely due to the inadequate sample of eimerian molecular data available for reliable phylogenetic analyses. In this study, we extend the data set by adding 71 new sequences of coccidia infecting 16 small-mammal genera, mostly rodents. According to the respective feasibility of PCR gene amplification, the new samples are represented by one or more of the following genes: nuclear 18S rRNA, plastid ORF 470, and mitochondrial COI. Phylogenetic analyses of these sequences confirm the previous hypothesis that Eimeria, in its current morphology-based delimitation, is not a monophyletic group. Several samples of coccidia corresponding morphologically to other genera are scattered among the Eimeria lineages. More importantly, the distribution of eimerians from different hosts indicates that the clustering of eimerian species is influenced by their host specificity, but does not arise from a cophylogenetic/cospeciation process; while several clusters are specific to a particular host group, inner topologies within these clusters do not reflect host phylogeny. This observation suggests that the host specificity of Eimeria is caused by adaptive rather than cophylogenetic processes.     

Description of a new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from the western Derby eland Taurotragus derbianus derbianus Gray (Artiodactyla: Bovidae) in Senegal

Examination of faecal samples from semi-captive western Derby elands Taurotragus derbianus derbianus Gray, in the Bandia and Fathala Reserves of Senegal, revealed the presence of o?cysts of the genus Eimeria Schneider, 1875 that we considered to represent a new species, Eimeria derbiani n. sp. The new species possesses nearly ellipsoidal o?cysts (length/width ratio 1.3) with a bi-layered wall and an average size of 27.6 × 21.5 μm. E. derbiani possesses a micropyle covered by a micropylar cap and ovoidal, single-layered sporocysts with an average size of 14.9 × 7.7 μm, each with a Stieda body. Sporozoites of E. derbiani possess a large refractile body and a nucleus. Sporulation lasted for 2 days at 23°C. The new species is differentiated from the two species parasitising Taurotragus oryx Pallas, E. canna Triffitt, 1924 and E. triffittae Yakimoff, 1934.     

Molecular characterization and phylogenetic analysis of Eimeria from turkeys and gamebirds: implications for evolutionary relationships in Galliform birds

In order to determine the evolutionary relationships among Eimeria species that parasitize birds of the Galliformes, the 18s rDNA gene and a portion of the cytochrome oxidase subunit 1 (cox-1) were amplified from Eimeria species isolated from turkeys, chukars, and pheasants. The phylogenetic analysis of these sequences suggests that species infecting chickens are polyphyletic and, therefore, do not all share a direct common ancestor. Both the 18s rDNA and the cox-1 sequences indicate that Eimeria tenella and Eimeria necatrix are more closely related to Eimeria of turkeys and pheasants than to other species that infect the chicken. It is, therefore, likely that the chicken Eimeria spp. represent 2 separate ancestral colonizations of the gut, one of which comprises E. tenella and E. necatrix that infect the ceca, while the other includes Eimeria acervulina, Eimeria brunetti, Eimeria maxima, and Eimeria mitis, which infect the upper regions of the intestine.     

Description and phylogeny of a new species of Eimeria from double-crested cormorants (Phalacrocorax auritus) near Fort Gaines, Georgia

The renal parasite Eimeria auritusi has caused several mortality events in double-crested cormorants (DCC; Phalacrocorax auritus) in the Midwest and southeastern United States. This parasite has only been detected during large-scale outbreaks, and its presence and prevalence in healthy populations of cormorants is unknown. In this study, 80 DCC were collected from the Chattahoochee River near Fort Gaines, Georgia, and examined for kidney and intestinal coccidia. Eighteen (22.5%) and 56 (70%) of the DCC were positive for E. auritusi and a new species of intestinal Eimeria, respectively. Oocysts of the new intestinal Eimeria species had a thin colorless wall, were ovoid with rare bumps on the outer surface, and measured 17.1 microm +/- 1.5 x 14.7 microm +/- 1.0 (16-18.5 x 13-17), with an average length:width ratio of 1.17 microm (1.03-1.29). A prominent micropyle (4-4.5 microm) was present, and a large oval-to-round polar body (2.5 microm) was located beneath the micropyle. Sporocysts were ovoid and measured 9.6 microm +/- 0.6 x 5.9 microm +/- 0.5 (8.5-10.5 x 5-6.5), with an average length:width ratio of 1.63 (1.3-1.82) with small stieda body present. Amplification and sequencing of a fragment of the 18S rRNA gene indicated that the 2 DCC Eimeria species and 2 Eimeria species from cranes were in a separate group from other Eimeriidae. These data indicate that E. auritusi and this new species of intestinal Eimeria are prevalent in this apparently healthy DCC population. The cause of renal coccidiosis outbreaks in other populations of cormorants is unknown but could be due to crowding or stress during the winter months or some other associated pathogen or immunosuppressor that might predispose individuals to clinical disease.     

Morphological and molecular characterization of a novel eimerian species (Apicomplexa: Eimeriidae) from the Australian pelican Pelecanus conspicillatus Temminck, 1824 (Pelecaniformes: Pelecanidae) in Western Australia

A new Eimeria Schneider, 1875 species is described from an Australian pelican Pelecanus conspicillatus Temminck, 1824 in Western Australia. Sporulated oocysts (n = 23) subspheroidal, 33–35 × 31–33 (34.1 × 32.0) μm; length/width (L/W) ratio 1.0–1.1 (1.07). Wall bi-layered, 1.2–1.5 (1.4) μm thick, outer layer smooth, c.2/3 of total thickness. Micropyle absent, but 2 or 3 polar granules surrounded by a thin membrane, apparently residual, are present. Sporocysts (n = 23) elongate ellipsoidal or capsule shaped, 19–20 × 5–6 (19.5 × 5.6) μm; L/W ratio 3.4–3.8 (3.51). Stieda body vestigial and barely discernible, 0.5 × 1.0 μm; sub-Stieda and para-Stieda bodies absent; sporocyst residuum present, composed of a few dense spherules dispersed among the sporozoites. Sporozoites with robust anterior and posterior refractile bodies and centrally located nucleus. Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene. At the 18S locus, the new isolate shared 98.6% genetic similarity with Eimeria fulva Farr, 1953 (KP789172), which was identified from a goose in China. At the 28S locus, the new isolate shared the highest similarity of 96.2% with Eimeria hermani Farr, 1953 (MW775031) identified from a whooper-swan (Cygnus cygnus (Linnaeus, 1758)) in China. At the COI gene locus, this new isolate was most closely related to Isospora sp. isolate COI-178 and Eimeria tiliquae [25,26], presented 96.5% and 96.2% genetic similarity, respectively. Based on the morphological and molecular data, this isolate is a new species of coccidian parasite, which is named Eimeria briceae n. sp.     

Accurate analysis of prevalence of coccidiosis in individually identified wild cranes in inhabiting and migrating populations in Japan

Eimeria gruis and E. reichenowi cause coccidiosis, a major parasitic disease of cranes. By non-invasive molecular approaches, we investigated the prevalence and genetic characterization of pathogens in two Japanese crane habitats; one is Hokkaido inhabited by the endangered red-crowned crane, and the other is Izumi in Kyushu where populations that consist mainly of vulnerable hooded and white-naped cranes migrate in winter. The non-invasively collected faecal samples from each wintering population were first subjected to host genomic DNA-targeted analyses to determine the sample origin and avoid sample redundancy. Extremely high prevalence was observed in the Izumi populations (> 90%) compared with the Hokkaido population (18-30%) by examining 470 specimens by microscopy and PCR-based capillary electrophoresis (PCR-CE), using genetic markers in the second internal transcribed spacer (ITS2). Correspondence analysis of PCR-CE data revealed differences in community composition of coccidia between hooded and white-naped cranes. 18S rRNA and ITS2 sequences were determined from single oocysts excreted by red-crowned and hooded cranes. Phylogenetic analysis of 18S rRNA suggested that E. reichenowi was polyphyletic while E. gruis was monophyletic. Together with PCR-CE data, these results indicate different host specificity among the E. reichenowi type. Our data suggest that E. reichenowi comprises multiple species.     

Five new species of Coccidia (Apicomplexa: Eimeriidae) from colubrid snakes of Ecuador.

Fecal samples from 11 colubrid snakes, representing 10 species, collected in Ecuador during October 1994 were examined for coccidian parasites. Feces of 4 individuals, representing 4 host species, contained coccidian oocysts. Three species of Eimeria and 2 species of Isospora were observed and are described here as new. Oocysts of both Eimeria and Isospora were found in the feces of a slug-eating snake, Dipsas vermiculata. Sporulated oocysts of the Eimeria sp. are spheroid to subspheroid, 16.7 by 16.6 microm (14-18 by 14-18 microm) and those of the Isospora sp. are spheroid and 15.0 microm (13-18 microm) in diameter. Imantodes cenchoa, the common bluntheaded treesnake, was infected with a species of Eimeria. These sporulated oocysts are ellipsoid, 23.3 by 16.2 microm (25-21 by 15-17 microm). Sporulated eimerian oocysts from Leptodeira annulata, the southern cat-eyed snake, are subspheroid, 22.5 by 18.8 microm (19-26 by 17-21 microm). Feces of a juvenile Imantodes lentiferus, the bluntheaded vine snake, contained ovoid to ellipsoid isosporan oocysts, which measured 21.6 by 15.0 microm (20-23 by 14-16 microm) when sporulated.     

DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)

Partial (～ 780 bp) mitochondrial cytochrome c oxidase subunit I (COI) and near complete nuclear 18S rDNA (～ 1,780 bp) sequences were directly compared to assess their relative usefulness as markers for species identification and phylogenetic analysis of coccidian parasites (phylum Apicomplexa). Fifteen new COI partial sequences were obtained using two pairs of new primers from rigorously characterised (sensu Reid and Long, 1979) laboratory strains of seven Eimeria spp. infecting chickens as well as three additional sequences from cloned laboratory strains of Toxoplasma gondii (ME49 and GT1) and Neospora caninum (NC1) that were used as outgroup taxa for phylogenetic analyses. Phylogenetic analyses based on COI sequences yielded robust support for the monophyly of individual Eimeria spp. infecting poultry except for the Eimeria mitis/mivati clade; however, the lack of a phenotypically characterised strain of E. mivati precludes drawing any firm conclusions regarding this observation. Unlike in the 18S rDNA-based phylogenetic reconstructions, Eimerianecatrix and Eimeria tenella formed monophyletic clades based on partial COI sequences. A species delimitation test was performed to determine the probability of making a correct identification of an unknown specimen (sequence) based on either complete 18S rDNA or partial COI sequences; in almost all cases, the partial COI sequences were more reliable as species-specific markers than complete 18S rDNA sequences. These observations demonstrate that partial COI sequences provide more synapomorphic characters at the species level than complete 18S rDNA sequences from the same taxa. We conclude that COI performs well as a marker for the identification of coccidian taxa (Eimeriorina) and will make an excellent DNA 'barcode' target for coccidia. The COI locus, in combination with an 18S rDNA sequence as an 'anchor', has sufficient phylogenetic signal to assist in the resolution of apparent paraphylies within the coccidia and likely more broadly within the Apicomplexa.     

利用叶绿体DNA trnL-F序列初步探讨菊科风毛菊属的系统发育

风毛菊属Saussurea DC.是菊科物种分化十分剧烈和分类处理十分困难的一个属。该属的单系起源性质、属下分类系统以及一些独特形态物种的系统位置尚不清楚,有待进一步验证。本文测定了代表该属5个亚属37种植物43个样品和川木香属Dolomiaea DC.的１种样品的叶绿体DNA trnL-F序列,并调取菜蓟族Cardueae Cass.与风毛菊属具有一定亲缘关系的13属的该序列,一起进行了分支分析,重点验证该属的属下形态分类系统以及形态特殊、青藏高原地区特有的雪兔子亚属subgen. Eriocoryne     

Phylogenetic and systematic study of Korean <Emphasis Type="Italic">Orius</Emphasis> species (Heteroptera: Anthocoridae) on the basis of molecular and morphological data

A phylogenetic and systematic study of Orius species (Heteroptera: Anthocoridae) from Korea has been conducted using both morphological and molecular characters. Thirty morphological character states were coded for 10 strains of 9 species. Five molecular markers, partial cytochrome c oxidase I (COI), cytochrome b (CytB), 16S rRNA (16S), 18S rRNA (18S), and 28S rRNA (28S), from mitochondrial and nuclear genes, were tested. Phylogenetic analyses based on molecular data were conducted by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic (BP) analyses. Analysis of morphological data was performed using the parsimony programs NONA, and the combined dataset of morphological and molecular data was analyzed using BP analyses. The results of this study indicate that use of COI and CytB enabled relatively effective identification of species, whereas the sequences of 16S, 18S and 28S did not enable identification of closely related species such as Orius minutus and O. strigicollis. We discuss the usefulness of the five molecular markers for determining phylogenetic relationships and identifying the species.     

Identification of a novel species of Eimeria Schneider, 1875 from the woylie,Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials

Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oöcysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O’Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O’Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O’Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O’Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O’Callaghan & O’Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.