Molecular and statistical approaches to the delimitation of Eimeriidae species: a case of extreme polymorphism in eimerian oocysts from the plumbeous pigeon Patagioenas plumbea (Vieillot, 1818) (Columbiformes) in South America.

The current work aimed to analyze, morphologically, statistically, and molecularly, oocysts shed from plumbeous pigeons, Patagioenas plumbea (Vieillot, 1818), from a locality at 2197 m of altitude near the Agulhas Negras peak, the highest point of the State of Rio de Janeiro, southeastern Brazil. The oocysts were extremely polymorphic, being subspheroidal, ovoidal, or ellipsoidal, in addition to having the random presence/absence of characteristic features associated with the oocyst wall, such as micropyle, micropyle cap, lateral micropyle, and outer veil/rough wall. Linear regression confirmed the extreme polymorphism of oocysts, showing that if all combinations of taxonomic characters in oocysts (morphotypes) were overestimated, 19 different species could be identified/described. In contrast, the means comparison analysis between oocysts with the presence/absence of characteristic features and the histograms showed equivalences and regularity in the distribution in the classes of measures, which indicate the presence of a single species in the measured oocysts. Molecular analyses were performed from the isolation of individual oocysts of different morphotypes, which had their genetic material extracted, amplified, and sequenced in 4 non-overlapping loci in the cox1 and cox3 genes and fragments of the small and large subunit rDNA of mitochondrial DNA. The sequences were 100% identical between the morphotypes, with the exception of a very small divergence observed at the locus that partially covers the cox3 gene. The phylogenetic analysis was inconclusive for the locus within the cox1 gene traditionally used for eimeriid coccidians; however, the other loci should have a promising future for phylogenetic studies when more sequences for the same genic regions are deposited in GenBank. Finally, the multifactorial analysis of the current work supported that the polymorphic oocysts shed from P. plumbea are a single species, which was named Eimeria patagioenasae, making this the twenty-second eimerian description from Columbiformes.

Molecular identification of Isospora coerebae Berto, Flausino, Luz, Ferreira amp; Lopes, 2010 (Chromista: Miozoa: Eimeriidae) from the bananaquit Coereba flaveola (Linnaeus, 1758) (Passeriformes: Thraupidae: Coerebinae) from Brazil.

Isospora coerebae Berto, Flausino, Luz, Ferreira Lopes, 2010 is a coccidian protozoan described from bananaquits Coereba flaveola (Linnaeus, 1758), on Marambaia Island, which is located on the southeastern Brazilian coast. In this current work, I. coerebae is identified from C. flaveola in a protected area close to Marambaia Island, but on the mainland, establishing a new location of parasitism, in addition to providing a preliminary genotypic characterization via sequencing of two regions of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. Its oocysts are sub-spherical, 24.4 22.9 m, with smooth, bilayered wall, ~1.7 m thick. Micropyle, polar granules and oocyst residuum are absent. Sporocysts are elongate ovoidal, 17.6 10.5 m. Stieda body prominent and rounded and sub-Stieda body short and wide. Sporocyst residuum is composed of scattered granules of different sizes. Sporozoites are vermiform with a prominent posterior refractile body. The oocysts of the current work are morphologically equivalent to the original description of I. coerebae, which have a typical and easily identifiable morphology, mainly in the Stieda and Sub-Stieda bodies. The two sequenced gene regions of the COI gene approximated I. coerebae to Isospora spp. from Southeastern Brazil, but also from Isospora spp. from passerines of North America, Europe and Asia. Although there is a small overlap between the two genic regions sequenced in the current work, it is estimated that the longer COI sequence, which was recently designed and still not widespread, should show better phylogenetic results in the future.

Isospora basileuterusi n. sp. (Apicomplexa: Eimeriidae) from the golden-crowned warbler Basileuterus culicivorus (Deppe) (Passeriformes: Parulidae) in South America.

Isospora basileuterusi Mello & Berto n. sp. is described based on material from the golden-crowned warbler Basileuterus culicivorus (Deppe) captured in the Itatiaia National Park (Parque Nacional do Itatiaia), a conservation unit in south-eastern Brazil. Oöcysts of the new species are ellipsoidal to ovoidal, measuring on average 25.2 × 21.1 µm, with a smooth, bi-layered wall, c.1.6 µm thick. Micropyle and oöcyst residuum are both absent, but one to three polar granules are present. Sporocysts are ellipsoidal to lemon-shaped, measuring on average 15.3 × 9.5 µm, with a knob-like Stieda body and a trapezoidal sub-Stieda body. Sporocyst residuum is present, usually as a body of membrane-bound granules. Sporozoites are vermiform, with refractile bodies. Four of the 19 warblers captured (21%) were infected with the new species. Molecular analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene revealed a similarity of 99.5% between the new species and Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries Serinus canaria (L.) in Western Australia. The oöcysts of I. basileuterusi n. sp. can be distinguished from the four other Isospora spp. recorded in hosts of the Parulidae, and from the molecularly most closely related species, by the typical ellipsoidal to lemon-shaped sporocysts, with small sub-Stieda body and a membrane-bound sporocyst residuum. Therefore, based on the morphological and molecular features, I. basileuterusi n. sp. is the fifth species described in a host of the family Parulidae and the first molecularly characterized via sequencing the cox1 gene.

Supplementary morphological data and molecular analyses of Eimeria labbeana (Labbé, 1896) Pinto, 1928 (Chromista: Miozoa: Eimeriidae) from columbiform birds in Portugal.

Columbiformes have a worldwide distribution, of which 166 species occur in Eurasia. They have been reported parasitized by coccidians recurrently in recent years; however, Eimeria labbeana (Labbé, 1896) Pinto, 1928, which is first Eimeria sp. from Columbiformes described in the late nineteenth century, is not taxonomically identified by its oocysts since the 1930s. In this context, the current study aimed to supplement the morphology of E. labbeana from Eurasian collared doves Streptopelia decaocto Frivaldszky, 1838 and from a common woodpigeon Columba palumbus Linnaeus, 1758 in Portugal, providing a preliminary genotypic characterization. Three of the four columbiforms were positive for oocysts identified as E. labbeana, which were morphologically revised as having micropyles, in addition to other minor adjustments. Oocysts from S. decaocto and C. palumbus were morphologically identical and equivalent in all morphometric aspects, besides having genotypic similarity of 99.5%. Phylogenetic analysis based on the mitochondrial cytochrome c oxidase subunit 1 gene resulted in a large clade with Eimeria spp. and Isospora spp. from different vertebrates and low similarity between Eimeria spp. from Columbiformes, whereas the phylogenetic analysis based on the small subunit ribosomal RNA gene resulted in well-supported monophyletic groups, including one with the coccidians of columbiform birds.

Distribution, redescription, and molecular identification of Isospora striata McQuistion et al. 1997 (Eimeriidae), from woodcreepers (Dendrocolaptidae) in South America.

Woodcreepers are passerines of the family Dendrocolaptidae, which have a high forest dependency. The current work aimed to redescribe Isospora striata McQuistion et al. 1997, from two new hosts in protected areas in Brazil, revealing new localities of parasitism, in addition to providing preliminary genotypic identifications via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from both host species. Isospora striata has oocysts that are subspheroidal to ovoidal, 19.4 × 16.8 µm with smooth wall. Oocyst residuum is absent, but micropyle and polar granules are present. Sporocysts are ovoidal, 13.6 × 8.3 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum is present and sporozoites with refractile body, nucleus, and striations. The morphological study and the 100% similarity in sequencing of the COI gene between samples of different dendrocolaptid species confirmed the identification of a single species, supporting the identification of I. striata in the Brazilian Atlantic forest and consequently the wide distribution of this coccidian species in the Neotropical Region.

Eimeria ferreirai n. sp. (Chromista: Miozoa: Eimeriidae) from doves Leptotila spp. (Columbiformes: Columbidae) from Brazil.

The doves and pigeons constitute a taxonomic group (Columbiformes: Columbidae) of granivorous and frugivorous birds with a worldwide distribution. The current work aims to describe morphologically and molecularly a new protozoan from white-tipped doves Leptotila verreauxi Bonaparte, 1855 and grey-fronted doves Leptotila rufaxilla (Richard Bernard, 1792) in Southeastern Brazil. Eimeria ferreirai n. sp. has oocysts that are sub-spherical to ellipsoidal, 21.4 × 18.8 µm, with smooth, bilayered wall, ~1.6 µm thick. Micropyle present. Oocyst residuum absent, but one to two polar granules are present. Sporocysts are elongate ovoidal to boomerang-shaped, 13.4 × 6.9 µm. Stieda body triangular to lozengal. Sporocyst residuum is composed of granules of different sizes. Sporozoites are vermiform with refractile body and nucleus. Sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the subsequent phylogenetic molecular comparisons supported the description of the new species, since the maximum similarity was 90-95% with eimeriid species of Columbiformes, Anseriformes, Galliformes and Passeriformes. Thus, this is the first coccidian species reported from Leptotila spp. and the twentieth description of an eimerian from Columbiformes in the World.

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.

The Use of Oral Beta-Blockers and Clinical Outcomes in Patients with Non-ST-Segment Elevation Acute Coronary Syndromes: a Long-Term Follow-Up Study.

BACKGROUND: The role of beta-blockers in patients with acute coronary syndromes is mainly derived from studies including patients with ST-segment elevation myocardial infarction. Little is known about the use of beta-blockers and associated long-term clinical outcomes in patients with non-ST-elevation acute coronary syndromes (NSTEACS). METHODS: We analyzed short- and long-term clinical outcomes of 2921 patients with NSTEACS using or not oral beta-blockers in the first 24 h of the acute coronary syndromes (ACS) presentation. The association between beta-blocker use and mortality was assessed using a propensity score adjusted analysis (N = 1378). RESULTS: Patients starting oral beta-blockers in the first 24 h of hospitalization, compared with patients who did not, had lower rates of in-hospital mortality (OR = 0.52, 95% CI 0.33 to 0.74, P = 0.002) and higher mean survival times in the long-term follow-up (11.86±0.4 years vs. 9.92±0.39 years, P < 0.001). CONCLUSION: The use of beta-blockers in the first 24 h of patients presenting with NSTEACS was associated with better in-hospital and long-term mortality outcomes.

Isospora sagittulae McQuistion & Capparella, 1992 (Apicomplexa: Eimeriidae) from antbirds (Passeriformes: Thamnophilidae) in the Amazon and Atlantic Forest of Brazil: with notes on its distribution and dispersion in the Neotropical region.

In the current study, Isospora sagittulae McQuistion and Capparella, 1992 (Protozoa: Apicomplexa: Eimeriidae) is reported from white-shouldered fire-eyes Pyriglena leucoptera (Vieillot, 1818) in the Atlantic Forest in southeastern Brazil. To date, this coccidian species was described from antbirds in Ecuador and Brazilian Amazon. In this sense, oocysts and measurements of the description of I. sagittulae from Amazonian antbirds were required from the deposit for comparison between samples from the Amazon and Atlantic Forest. The morphology was similar in all aspects, despite the polymorphism associated with the oocyst shape. DNA sequences for the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus of the oocysts had similarity of 100%. Therefore, these strong morphological, molecular, and ecological equivalences ensure the unique identification of I. sagittulae. Finally, this finding reveals the wide distribution of I. sagittulae in the Neotropical region and indicates that other antbirds in the Brazilian Cerrado should disperse I. sagittulae to the Amazon and Atlantic Forest.

Eimeria spp. from Japanese quails (Coturnix japonica): new characteristic features and diagnostic tools / Eimeria spp. de codornas japonesas (Coturnix japonica): novas características e ferramentas de diagnóstico

The Japanese quail Coturnix japonica originated from North Africa, Europe and Asia, is used worldwide as an experimental animal and model for aviculture. The current paper characterizes Eimeria bateri, Eimeria tsunodai and Eimeria uzura recovered from C. japonica. Based on the fact that quails have a global distribution, as are their coccidia, the findings of this study should provide the means for diagnosis of those Eimeria spp. in other regions and continents. Eimeria bateri showed the greatest intensity of infection and shed oocysts from the fourth day after infection; in contrast, E. tsunodai and E. uzura shed oocysts from the fifth day after infection. The three species shared a high degree of similarity and were all polymorphic. Yet, the application of line regressions, histograms and ANOVA provided means for the identification of these species. Finally, the algorithm was very efficient since verified that resultant values were not superimposed.

A codorna japonesa Coturnix japonica originária do norte da África, Europa e Ásia, é utilizada mundialmente como um animal experimental e modelo para avicultura. O presente trabalho caracteriza Eimeria bateri, Eimeria tsunodai e Eimeria uzura recuperadas de C. japonica. Baseado no fato de que as codornas têm uma distribuição global, como são os seus coccídios, os resultados deste estudo devem propiciar o diagnóstico destas Eimeria spp. em outras regiões e continentes. Eimeria bateri demonstrou a maior intensidade de infecção e eliminaram oocistos a partir do quarto dia após infecção, em contraste E. tsunodai e E. uzura eliminaram oocistos a partir do quinto dia após infecção. As três espécies foram morfometricamente semelhantes e polimórficas. No entanto, a aplicação da regressão linear, histogramas e ANOVA proveram meios para a identificação destas espécies. Finalmente, o algoritmo foi totalmente eficiente uma vez que valores resultantes não foram sobrepostos.