Phylogenomics Identifies a New Major Subgroup of Apicomplexans, Marosporida class nov., with Extreme Apicoplast Genome Reduction.

The phylum Apicomplexa consists largely of obligate animal parasites that include the causative agents of human diseases such as malaria. Apicomplexans have also emerged as models to study the evolution of nonphotosynthetic plastids, as they contain a relict chloroplast known as the apicoplast. The apicoplast offers important clues into how apicomplexan parasites evolved from free-living ancestors and can provide insights into reductive organelle evolution. Here, we sequenced the transcriptomes and apicoplast genomes of three deep-branching apicomplexans, Margolisiella islandica, Aggregata octopiana, and Merocystis kathae. Phylogenomic analyses show that these taxa, together with Rhytidocystis, form a new lineage of apicomplexans that is sister to the Coccidia and Hematozoa (the lineages including most medically significant taxa). Members of this clade retain plastid genomes and the canonical apicomplexan plastid metabolism. However, the apicoplast genomes of Margolisiella and Rhytidocystis are the most reduced of any apicoplast, are extremely GC-poor, and have even lost genes for the canonical plastidial RNA polymerase. This new lineage of apicomplexans, for which we propose the class Marosporida class nov., occupies a key intermediate position in the apicomplexan phylogeny, and adds a new complexity to the models of stepwise reductive evolution of genome structure and organelle function in these parasites.

Parasites in a hotspot: diversity and specificity patterns of apicomplexans infecting reptiles from the Socotra Archipelago.

Although parasites represent a major component of biodiversity, they remain poorly assessed, especially in remote regions. In this study, we screened 461 reptiles from Socotra, the largest and most biologically diverse archipelago in Arabia. Using 18S rRNA primers, we detected various apicomplexan parasites, namely haemogregarines, sarcocystids and eimeriids. Haemogregarines were the most common and genetically diverse, followed by sarcocystids (genus Sarcocystis) and eimeriids (genera Isospora and Lankesterella). All were related to parasites of other reptiles, including species from Arabia, Northern Africa and Asia. Like their 29 endemic reptile hosts, almost all Socotran parasites presented high genetic divergence and ecological differences from those found elsewhere, and probably represent undescribed endemic species. Among hosts, skinks were the most parasitized, which contrasted with similar studies from other areas, probably due to their more generalist diet and habitat use. As expected due to its high species richness, geckos harboured the highest parasite diversity in the archipelago. Parasite diversity also seemed to be correlated to island size, as the largest island harboured most haplotypes. This study emphasizes the importance of screening parasites in wild hosts from remote regions and of considering host ecology to understand disease transmission across taxa.

A novel coccidian (Apicomplexa: Eimeriidae) from Scotophilus leucogaster (Chiroptera: Vespertilionidae) in southern Saudi Arabia.

A novel species of coccidia, resembling a member of the genus Eimeria, was found in bats, Scotophilus leucogaster, collected in southern Saudi Arabia has been described on the basis of unsporulated oocysts and DNA sequencing of the Internal Transcribed Spacer 1 (ITS1) and partial 18S rDNA regions. Unsporulated oocysts of this form are ovoidal to spheroidal and had a 2-layered wall, 1.5-2.0 (1.9 ± 0.2); the outer layer was light blue with striations, and thicker than the inner, darker layer. No micropyle was present. Unsporulated oocysts (N = 150) measured 27.2 × 22.1 (25-30 × 20-25), length width ratio, 1.2 (1.1-1.4). There was no evidence of an oocyst residuum and/or polar granule. This parasite was detected in 2/7 (29%) S. leucogaster collected from southern Saudi Arabia. Oocysts incubated at 25 °C in 2.5% K2Cr2O7 did not sporulate after > 1 month. Unsporulated oocyst measurements were compared with other coccidian parasites of bats that discharge oocysts in their feces. Sequences of the ITS1 and the 18S rDNA regions obtained from the unsporulated oocysts grouped this coccidium from S. leucogaster with eimerian species from various rodent and squirrel species. It is critical that future investigators obtain fully sporulated oocysts of this coccidium for full description of the parasite recovered in our study so it can be correctly assigned to genus and given an accurate binomial.

Effects of Goussia infecting the blue whiting and phylogenetic placement of Goussia infecting marine fish off Northern Portugal.

Coccidian parasites of fish have received considerably less attention than their terrestrial counterparts, and within piscine hosts, most studies have focused on freshwater fish. The present study aimed to describe oocyst morphology, phylogenetic affinities, and the impacts of coccidian parasites infecting the internal organs of a commercially valuable marine fish, the blue whiting (Micromesistius poutassou), captured off the Portuguese coast. As part of the phylogenetic analysis, sequences from coccidians infecting the pout (Trisopterus luscus) and the Atlantic chub mackerel (Scomber colias) were included, and the oocyst morphology of the coccidians infecting the former was also reported. Results showed that the prevalence of coccidiosis in the blue whiting was very high (> 82%), occurring in all analyzed organs, despite being more abundant in the liver. A significant negative correlation was found between the abundance of the parasites in the liver and host condition index (p < 0.05), which indicates a negative effect on the fitness of this host. Phylogenetic analyses of the parasites found in all three species examined identified three different species of Goussia, closely related to Goussia clupearum. Adding to previous research, we propose the existence of a fourth group of Goussia, the clupearum type, able to infect multiple organs and phylogenetic related with G. clupearum.

Full Mitochondrial Genome and Nuclear 18S rDNA Sequences Refine the Taxonomic Placement of Choleoeimeria taggarti n. comb. from the Prostate of Antechinus flavipes (Yellow-Footed Antechinus).

An unusual coccidian parasite was described previously from the prostate of a male Antechinus flavipes (family: Dasyuridae; common name: yellow-footed antechinus). Morphometrics and a partial nuclear 18S small subunit rDNA (18S rDNA) sequence were used to assign this parasite to the genus Eimeria; it was named Eimeria taggarti. We generated full nuclear 18S rDNA and mitochondrial genome sequences from this parasite and used the newly completed 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) sequences to perform a more in-depth phylogenetic analysis. The parasite clustered closely with Choleoeimeria spp. and Acroeimeria spp. infecting herptiles in a well-supported clade that was the sister lineage to the Eimeriidae sensu stricto. The mitochondrial genome of this parasite contained 2 inverted segments compared to mitochondrial genomes from parasites in the Eimeriidae sensu stricto (i.e., Stieda body-possessing coccidia with 4 dizoic sporocysts); this mitochondrial genome arrangement was shared with the only Choleoeimeria species for which sequence data were available publicly. Examination of histological preparations and TEM images uncovered bivalvate sporocysts and otherwise confirmed previously described morphological features of the parasite. Based on our phylogenetic analyses and histological observations, we propose the generic reclassification of E. taggarti to Choleoeimeria taggarti n. comb.

Coccidia of Columbiformes: a taxonomic review of its Eimeriidae species and Eimeria columbinae n. sp. from Columbina talpacoti (Temminck, 1809) from Brazil.

Coccidia (Chromista: Miozoa: Eimeriidae) of columbiform birds (Aves: Columbiformes) have been described since the end of the nineteenth century; however, some of these descriptions were poorly detailed or inconclusive. In this sense, the current work makes a detailed taxonomic revision reconsidering and organizing 18 Eimeria spp. and two Isospora spp. previously described or reported of Columbiformes. Along with this, a new species of Eimeria is morphologically and molecularly identified by the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and by the 18S small subunit ribosomal RNA (18S) gene from the ruddy ground-dove Columbina talpacoti (Temminck, 1809) in the Médio Paraíba region of the State of Rio de Janeiro, southeastern Brazil. Eimeria columbinae n. sp. has subspheroidal oocysts, 14.7 × 13.2 µm, with smooth, bi-layered wall, ~ 1.1 µm and length/width ratio of 1.1. Micropyle and oocyst residuum are present, but polar granule is absent. Sporocysts are ellipsoidal to slightly asymmetrical, 9.0 × 5.1 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum present and sporozoites with refractile body and nucleus. This is the 19th description of an eimerian from Columbiformes in the World, and the second to have a molecular identification of the COI and 18S genes.

Gastric coccidiosis in Thoracocharax stellatus caused by Goussia guamaensis n. sp. (Apicomplexa: Eimeriidae) from the Amazon region.

Ninety-seven specimens of spotfin hatchetfish, Thoracocharax stellatus, an ornamental freshwater species from the Amazon basin, were captured in the basin of the Guamá River in the municipality of Belém, in northern Brazil, and analysed for coccidiosis infection. Overall, 26 of the specimens were infected by apicomplexan parasites of the genus Goussia, with unsporulated forms being found in the gastric epithelium and sporulated forms in the intestinal lumen. The spheroid oocysts (mean diameter: 13.2 ± 1.7 µm) have four elliptical sporocysts. A partial sequence of the SSU rDNA of the new species was obtained, which contained 1,121 base pairs, with 43.8% guanine + cytosine (G + C), and the bases distributed as follows: A = 28.1%, C = 18.3%, G = 25.5% and T = 28.1%. The combined analysis of the morphometric and phylogenetic evidence confirmed that the specimens represented the genus Goussia and were allocated to a new species, Goussia guamaensis n. sp., which is described here.

Newly Described Coccidia Goussia bayae from White Perch Morone americana: Morphology and Phylogenetics Support Emerging Taxonomy of Goussia within Piscine Hosts.

In March and April 2016, 150 white perch ( Morone americana) were collected from various localities in Chesapeake Bay and examined for coccidia. A previously undescribed species of coccidia was observed in the hepatic bile ducts and gallbladder of all white perch (100%) examined. We describe this species using morphological characteristics, histology, and gene sequences of the small-subunit ribosomal DNA ( rDNA), large-subunit rDNA, and mitochondrial genes cytochrome oxidase 1 ( COI), cytochrome oxidase b ( Cytb), and cytochrome oxidase 3 ( COIII). Oocysts of Goussia bayae n. sp. were subspherical with a single-layered smooth wall and measured (length [L] × width [W]) 26.2 × 21.8 µm, with a L/W ratio of 1.2. A micropyle was present but a micropyle cap, polar granules, and oocyst residuum were absent. Each oocyst contained 4 sporocysts that were ellipsoidal and measured (L × W) 12.6 × 7.8 µm, with a L/W ratio of 1.6. A pair of sporozoites was present, but sporocysts lacked a Stieda body and residuua. Meronts and gamonts were epicellular in biliary epithelial cells and oocysts were coelozoic in hepatic and common bile ducts and gallbladder. This is the first report of Goussia spp. from white perch and the first mitochondrial DNA sequence reported from a Goussia species. Phylogenetic analysis indicates basal placement of G. bayae to Eimeriidae, Choleoeimeria, and Sarcocystidae.

Intranuclear coccidiosis in tortoises - discovery of its causative agent and transmission.

Intranuclear coccidiosis of testudines (known as TINC) is an emerging disease in chelonians. Although endogenous stages were repeatedly detected in various tissues, attempts to find the oocysts in faeces failed, leaving the question of the transmission and classification of the causative agent of TINC unresolved. We recorded small spherical oocysts (∼6-7 µm in diameter) of an eimeriid coccidium in faeces of a leopard tortoise (Stigmochelys pardalis). Sporulated oocysts were used for the experimental oral inoculation of juvenile coccidia-free tortoises representing 5 species (S. pardalis, Testudo graeca, T. hermanni, T. horsfieldii, and Geochelone sulcata). The oocysts' association with TINC was confirmed based on clinical signs, histopathological findings of intranuclear endogenous stages of the coccidium in many organs (including intestine), and by the partial 18S rDNA sequence analysis of the DNA isolated from organs of the experimentally infected animals and from a single naturally infected as well as from all experimentally infected tortoises. Breeding colonies of chelonians should be screened for this pathogen in order to prevent its further spread and unwanted introduction into endangered free-ranging chelonian populations.

Pathological and molecular characterization of systemic isosporosis (atoxoplasmosis) in captive green-winged saltator (Saltator similis).

Systemic isosporosis, also called atoxoplasmosis or visceral coccidiosis, is a disease that affects birds in general. Pathogenesis of systemic isosporosis and its etiologic agent have not been well characterized, but taxonomically Atoxoplasma is currently considered a junior objective synonym of Isospora. The present report aimed to describe pathological and molecular findings of systemic isosporosis in captive green-winged saltators (Saltator similis) from the State of Espírito Santo, Brazil. In a commercial breeding facility eleven birds with two to nine months of age died from 2015 to 2016. These birds developed nonspecific clinical signs, including bristly feathers, hyporexia, loss of weight, and apathy. Two birds were necropsied, and grossly there were hepatomegaly, splenomegaly, necrosis of lymphoid follicles, hepatic necrosis, and severe enteritis. Merozoites were observed in the heart, small intestine, proventriculus, brain, liver, spleen, and kidneys. 23 S RNA PCR amplicons from DNA extracted from the liver and the intestinal contents had 99% identity with Atoxoplasma sp., whereas amplicons of mitochondrial cytochrome c oxidase subunit 1 ha d 97% identity with Isospora greineri. In conclusion, this report indicates that systemic isosporosis in green-winged saltator is a disease that affects the spleen, liver, and small intestine, with high mortality for young birds, resulting in significant loses to commercial breeding facilities.

Cloning, expression and molecular characterization of a Cystoisospora suis specific uncharacterized merozoite protein.

BACKGROUND: The genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico. METHODS: The 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively. RESULTS: The CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite. CONCLUSIONS: The results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.

Redescription and new locality record of Choleoeimeria turcicus from the gallbladder of Hemidactylus turcicus in Egypt, with notes on its endogenous development.

Upton et al. J Protozool 35:24-25, 1988 originally described Eimeria turcicus from the gallbladder of the Mediterranean gecko, Hemidactylus turcicus, in the USA. Shortly after establishing the genus Choleoeimeria, Paperna and Landsberg S Afr J Zool 24:345-355, 1989 transferred E. turcicus to this genus, renaming it as Choleoeimeria turcicus. This paper reports the findings of a survey of coccidian parasites of lizards in Egypt, during which tetrasporocystic oocysts were reported from the gallbladder of H. turcicus. Based on the oocysts' morphological and morphometric characteristics and the same host, it can be deduced for the first time that the present species is conspecific with E. turcicus. In the present study, the gallbladder was the sole site for the endogenous development and no endogenous stages were noticed in the intestine. The endogenous stages induce hypertrophy and displacement of the infected cells from the original biliary epithelium to form a prolonged and branching outgrowth. We therefore followed Paperna and Landsberg S Afr J Zool 24:345-355, 1989 and considered the biliary coccidium recorded in this study to be a member of the genus Choleoeimeria. This represents a new locality record for C. turcicus, which was previously known in the USA and Israel.

Protozoal nodular dermatitis and panniculitis in a Rottweiler puppy caused by Caryospora bigenetica.

BACKGROUND: Caryospora bigenetica is an intracellular protozoan parasite in snakes and raptors (primary hosts) and rodents (secondary host). Experimental infection has been documented in mice, pigs and goats; natural infection in dogs is rare. OBJECTIVES: To describe the clinical presentation, histological features, treatment and outcome of a case of protozoal nodular dermatitis and panniculitis in a Rottweiler puppy caused by C. bigenetica. RESULTS: The puppy presented with generalized subcutaneous nodules measuring up to 2 cm in diameter. Histopathology revealed marked suppurative to pyogranulomatous dermatitis and panniculitis with intralesional protozoal organism. PCR and DNA sequencing confirmed infection with C. bigenetica. Treatment with a combination of oral trimethoprim-sulfamethoxazole (TMS), pyrimethamine and high-dose clindamycin (20 mg/kg twice daily) resulted in resolution of lesions in 6 weeks. Discontinuation of the treatment 2 weeks later was followed by a rapid relapse of skin lesions. Clindamycin and TMS were restarted and all lesions resolved within 2 weeks; TMS was discontinued 4 weeks later due to adverse effects. The lesions remained in remission for 2 months while the puppy received clindamycin monotherapy before a second relapse of skin lesions occurred. CONCLUSION AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first documentation of the treatment and outcome of C. bigenetica cutaneous infection in a dog. Although remission of clinical signs can be achieved with combination therapy of clindamycin and TMS, long-term management is challenging and relapses should be anticipated.

The complete mitochondrial genome of Caryospora bigenetica (Eimeriidae, Eucoccidiorida, Coccidiasina, Apicomplexa).

The 6313 bp complete mitochondrial (mt) genome of Caryospora bigenetica was sequenced directly from PCR products. The mt genome was comparable in size, gene content and order to those of other Eimeriid coccidia (e.g. Isospora or Eimeria species). Three protein-coding genes encoding COI, COIII and CytB were identified; numerous rDNA fragments (19 LSU and 14 SSU) were interspersed among the CDS. Nucleotide composition was A + T biased (66%). The mitochondrial genomes of Eimeriid coccidia appear to share the same gene order and content; mt genome sequences can provide molecular data useful for diagnostics, taxonomy and phylogenetic relationships of Eimeriid coccidia.

Morphological and molecular characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa: Eimeriidae, 1989, Paperna and Landsberg) in a western bearded dragon (Pogona minor minor).

A new species, Choleoeimeria pogonae n. sp. is described from a Western bearded dragon (Pogona minor minor) in Western Australia. Sporulated oocysts (n = 48) were cylindroidal in shape. Oocyst length, 27.0 (26.0-28.3) µm, oocyst width, 15.2 (14.0-16.5) µm, oocyst length/width ratio (L/W) 1.8 (1.6-1.9), each with 4 sporocysts (Eimeria-like) and a polar granule, but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal in shape, sporocyst length, 10.0 (9.0-11.0) µm, sporocyst width 8.5 (7.0-9.5) µm, sporocyst L/W ratio, 1.2 (1.1-1.3). Stieda, substieda and parasubstieda bodies were all absent. Molecular analysis was conducted at the 18S rRNA and cytochrome c oxidase I (COI) loci. Phylogenetic analysis of 18S sequences revealed that C. pogonae n. sp. grouped together with another four Choleoeimeria spp. and exhibited 99.1%-99.4% genetic similarity. At the COI locus, C. pogonae n. sp. was in an independent clade and had the highest similarity (80.4%) to Eimeria cf. mivati from a chicken (Gallus gallus domesticus). According to the morphological and molecular data, this isolate is a new species of coccidian parasite. This study further supports the taxonomy of Choleoeimeria spp. as a new genus based on molecular phylogenetic analysis.

A new Caryospora coccidian species (Apicomplexa: Eimeriidae) from the laughing kookaburra (Dacelo novaeguineae).

A new Caryospora coccidian species is described from the laughing kookaburra (Dacelo novaeguineae). Sporulated oocysts (n=30) are ovoid in shape with a smooth, colourless, bilayered oocyst wall and measure 31.4×29.3 (30.0-32.0×28.0-31.0) µm with a shape index of 1.1. Oocysts contain one spheroidal to subspheroidal sporocyst, 21.2×20.6 (20.0-24.0×20.0-21.0) µm. A spheroidal shaped sporocyst residuum is present; micropyle, Stieda, substieda and parastieda bodies are absent. Vermiform sporozoites (n=8) are arranged either parallel or randomly in the sporocyst, measuring 17.0×4.8 (16.0-18.0×4.0-6.0) µm, with a L/W ratio of 3.5. There is a large spheroidal, posterior refractile body in the middle of the sporozoite. Morphologically, this new species is most similar to Caryospora. The prevalence of this parasite was 6.7% in birds sampled in the morning and 33.3% from those sampled after midday. Further molecular characterisation was conducted at two loci; the 18S and 28S ribosomal RNA (rRNA). At the 18S locus, the new species of Caryospora was most closely related to Besnoitia besnoiti (99.2% similarity) and Hammondia triffittae (98.8% similarity). Although, no 28S partial sequences from Caryospora were available in GenBank, the highest similarity was with B.besnoiti (91.3%). Based on morphological and molecular data, this coccidian parasite is a new species that to date has not been reported. The new coccidian parasite is named Caryospora daceloe n. sp. after its host D. novaeguineae (the laughing kookaburra).

Coccidia species in endemic and native New Zealand passerines.

New Zealand native passerines are hosts to a large variety of gastrointestinal parasites, including coccidia. Coccidian parasites are generally host-specific, obligate intracellular protozoan parasites. In passerine birds, members of the genus Isospora are most common. Under natural conditions, these parasites seldom pose a threat, but stressors such as quarantine for translocation, overcrowding, or habitat changes may cause an infection outbreak that can severely affect wild populations. Although coccidia are important pathogens and have caused mortalities in kiwi (Apteryx spp.) and hihi (Notiomystis cincta), their prevalence, epidemiology, life cycles, and taxonomic relationships are still widely unknown in native New Zealand songbirds. Over a period of 3 years (2007-2009), we examined 330 fecal samples of six native passerine species: tui (Prosthemadera novaeseelandiae), North Island saddleback (Philesturnus carunculatus rufusater), North Island robin (Petroica longipes), silvereye (Zosterops lateralis), and fantail (Rhipidura fuliginosa). The overall prevalence by flotation of coccidian infection in the New Zealand bird species examined was 21-38 %, 21 % in North Island robin, 38 % in tui, and 25 % in saddleback. Similar to prior studies in other countries, preliminary sequencing results suggest that coccidia in passerines in New Zealand are members of the family Eimeriidae, unlike the phenotypically similar genus Cystisospora of mammals. Using molecular methods, we identified at least five new genetically distinct Isospora species in the examined birds (three in tui and one each in saddlebacks and North Island robins).

Coccidiosis in hihi/stitchbirds (Notiomystis cincta) due to coccidia of the Eimeriidae.

AIM: To describe the pathology of coccidiosis in hihi and to provide preliminary data on the taxonomy of the coccidia involved using molecular methods. METHODS: In an initial study from 1994 to 1997, gross and histopathological examinations were performed on 12 dead juvenile hihi from the National Wildlife Centre (NWC) at Mt. Bruce. In a second study during 2008-2010 DNA from sporulated oocysts and liver tissue was used for PCR analysis and sequencing. Faecal samples were also obtained from infected hihi from the NWC and examined for coccidial oocysts, which were then sporulated in the laboratory in 1994-1997 and 2007-2009. In addition, a post mortem was performed on a dead adult hihi from the NWC in 2008, and 18 archived hihi tissues from 11 individual birds stored at the Institute of Veterinary, Animal and Biomedical Sciences (IVABS) were used for DNA extraction. RESULTS: Severe gross and histopathological changes in the intestine and occasionally in the liver were found in the 12 dead birds examined. The morphological characteristics of the sporulated oocysts suggested that two types of coccidia were present. PCR analysis and sequencing of extracted DNA supported the existence of at least two different coccidia species in hihi. These were genetically more closely related to the genus Eimeria than to the morphologically similar genus Cystisospora (formerly Isospora) of mammals. In addition, one liver tissue sample that was examined post mortem was positive for at least two different coccidia species of the family Eimeriidae according to sequencing results, and the presence of extraintestinal coccidian stages was confirmed. CONCLUSIONS: Preliminary morphological and sequencing results suggest that two types of eimeriid coccidia are present and at least one of these commonly has extraintestinal stages. CLINICAL RELEVANCE: Coccidiosis in hihi is a serious disease capable of causing mortalities in juvenile and adult birds in captive situations. Treatment and control of the disease will be difficult as the extraintestinal stages of the organism are likely to be refractile to oral treatment.

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan.

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.

Goussia Labbé, 1896 (Apicomplexa, Eimeriorina) in Amphibia: diversity, biology, molecular phylogeny and comments on the status of the genus.

We provide new data on morphology, host specificity, life history, pathology and phylogeny of the coccidian genus Goussia in European anurans. Divergence in the SSU rDNA sequences (3-4%) of three Goussia isolates from three anuran hosts suggests that the isolates represent three distinct species. The isolate from Pelophylax kl. esculentus was determined as Goussia neglecta. The isolates from Rana dalmatina and Rana temporaria are considered conspecific and are, on the basis of host specificity, light microscopical, ultrastructural, and molecular phylogenetic data, described as a new species, Goussia noelleri. The new Goussia species from Bufo bufo remains unnamed. During the host's metamorphosis the infection disappears, however, oocysts are retained in the liver of post-metamorphic frogs. Molecular phylogenetic analysis showed that anuran-host Goussia form a well-supported monophyletic clade, which together with a clade represented by piscine Goussia metchnikovi, constitute basal lineages of the Eimeriidae. The two lineages show polytomy, likely reflecting undersampling of the basal eimeriid taxa. Goussia janae represents a distinct lineage, sister to the clade containing the other eimeriorinid taxa, suggesting a paraphyly of the genus Goussia. The identity of G. neglecta, the status of the genus Goussia, the presence of cryptic species in anuran-host Goussia and their ecological peculiarities are discussed.