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.

Detection of Toxoplasma gondii oocysts in soil and risk mapping in an island environment in the Northeast of Brazil.

Toxoplasmosis is an emerging and re-emerging infectious disease that can be transmitted through a contaminated environment. Environmental contamination is an emergency health issue, and determining its occurrence is fundamental to a One Health approach. In this study, we addressed the extent of environmental contamination and viability of Toxoplasma gondii oocysts in soil in different environments on Fernando de Noronha Island, Brazil. In addition, we performed species distribution modelling to predict the environmental suitability for coccidia persistence in the studied area. Soil samples were collected in 14 neighbourhoods of the Island and in the four main squares, creating a total of 95 soil samples (five samples per site). The samples were analyzed by the polymerase chain reaction (PCR) technique for the presence of the 18S ribosomal DNA gene of Apicomplexan protozoa, followed by genetic sequencing. We obtained 4.2% (4/95) positive soil samples with 100% similarity for T. gondii sequences. Two out of four positive sites on PCR showed viability of T. gondii oocysts through the mouse bioassay technique. As a result of the application of the species distribution modelling, environmental adequacy for the coccidia was observed throughout the Island. The results confirm the contamination of the soil in this insular environment by T. gondii oocysts and the environmental suitability by modelling application. These findings are an alert for the possibility of infection in animals and humans by contaminated soil, and for contamination of the maritime environment in addition to water resources for consumption by the local population.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Modifications of the U.S. food and drug administration validated method for detection of Cyclospora cayetanensis oocysts in prepared dishes: Mexican-style salsas and guacamole.

Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.

Polymorphism and genetic diversity of Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira & Lopes, 2015 (Eimeriidae) from antbirds (Thamnophilidae) in Brazil.

Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira & Lopes, 2015 was identified from a new host, the plain antvireo Dysithamnus mentalis (Temminck), and also from the white-shouldered fire-eye Pyriglena leucoptera Vieillot, in its type-locality, the Itatiaia National Park in the southeastern Brazil, and a preliminary genotypic characterisation by sequencing the mitochondrial cytochrome c oxidase subunit 1 gene is provided. The oöcysts recovered from P. leucoptera and D. mentalis were polymorphic and have genotypic differences that were not considered sufficient for the description of new species, but only different genotypes and morphotypes of I. parnaitatiaiensis related to each host. These morphological and molecular variations were associated with a process of ongoing speciation and in adaptive development to their respective host species.

Detection and genotypes of Toxoplasma gondii DNA in feces of domestic cats in Colombia.

The high prevalence of Toxoplasma gondii in the human population in Colombia has been linked to the existence of a high density of urban stray cats, exposing the whole population to a high density of oocysts. The goal of this study was to determine the DNA prevalence of T. gondii by conventional PCR and to phylogenetically analyze ROP18 sequences from positive samples in domestic cat (Felis catus) fecal samples in the city of Armenia, Quindío. Fecal samples from 140 cats were collected from 10 districts around the city. Samples were concentrated using Ritchie's method and analyzed through optical microscopy. Concentrates were used for DNA extraction followed by nested PCR amplification for T. gondii gene B1. PCR for ROP18 was performed on all B1 positive samples; the ROP18 sequences obtained were related to the Archetype I Brazilian and Chinese strains. No oocysts were detected by optical microscopy; however, 17.8% (25/140) B1 and 24% (6/25) ROP18 PCR-positive samples were detected. Phylogenetic analyses showed that isolates clustered into a single group. We assessed whether associations existed between T. gondii positive fecal samples and survey variables such as cat healthcare and socioeconomic characteristics of owners, but no statistically significant associations were found. The presence of T. gondii in cat feces is an important factor contributing to the high prevalence in the human population of this city.

TITLE: Détection d'ADN et génotypes de Toxoplasma gondii dans les fèces de chats domestiques en Colombie. ABSTRACT: La forte prévalence de Toxoplasma gondii dans la population humaine en Colombie a été liée à l'existence d'une forte densité de chats errants urbains, exposant l'ensemble de la population à une forte densité d'oocystes. Le but de ce travail était de déterminer la prévalence de l'ADN de T. gondii par PCR conventionnelle et d'analyser phylogénétiquement les séquences ROP18 d'échantillons positifs dans des échantillons fécaux de chat domestique (Felis catus) dans la ville d'Armenia, Quindío. Des échantillons fécaux de 140 chats ont été collectés dans 10 districts de la ville. Les échantillons ont été concentrés en utilisant la méthode de Ritchie et analysés par microscopie optique. Des concentrés ont été utilisés pour l'extraction d'ADN suivie d'une amplification par PCR nichée pour le gène B1 de T. gondii. La PCR pour ROP18 a été réalisée sur tous les échantillons positifs pour B1 ; les séquences ROP18 obtenues étaient apparentées aux souches Archétype I brésiliennes et chinoises. Aucun oocyste n'a été détecté par microscopie optique mais les échantillons étaient positifs par PCR pour 17,8 % (25/140) pour B1 et 24 % (6/25) pour ROP18. Les analyses phylogénétiques ont montré que les isolats formaient un seul groupe. Nous avons évalué s'il existait des associations entre des échantillons fécaux positifs à T. gondii et des variables d'enquête telles que les soins de santé des chats et les caractéristiques socioéconomiques des propriétaires, mais aucune association statistiquement significative n'a été trouvée. La présence de T. gondii dans les excréments de chats est un facteur important contribuant à la forte prévalence dans la population humaine de cette ville.

Cystoisospora felis infection in a captive jaguar cub (Panthera onca) in Michoacán, México.

Cystoisospora felis (Wenyon 1923) was identified in a 3-month-old, captive jaguar (Panthera onca) presenting with signs of gastrointestinal distress. The cub was fed beef, chicken, and commercial diet. Examination of fresh feces detected large (47.8 µm × 35 µm) unsporulated oocysts. Sporulated oocysts contained 2 sporocysts, each with 4 sporozoites. Oocyst morphometrics agreed with published features of C. felis described from domestic felines. Partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was PCR-amplified and sequenced; the resulting sequence showed 100% identity to a C. felis isolate from a domestic cat. This is the first molecularly confirmed report of C. felis infecting and producing clinically evident, enteric disease in a jaguar cub.

Morphological and molecular identification of Isospora massardi Lopes, Berto, Luz, Galvão, Ferreira & Lopes, 2014 (Chromista: Miozoa: Eimeriidae) from thrushes Turdus spp. (Passeriformes: Turdidae) in South America.

In the current work, Isospora massardi Lopes, Berto, Luz, Galvão, Ferreira & Lopes, [10] is identified from white-necked thrushes Turdus albicollis Vieillot, 1818, rufous-bellied thrushes Turdus rufiventris Vieillot, 1818 and from a new host, the yellow-legged thrush Turdus flavipes (Vieillot, 1818) in a new locality, the Itatiaia National Park, in Southeastern Brazil, providing a preliminary genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 18S small subunit ribosomal RNA genes. The oocysts and sporocysts of I. massardi of the current study are morphologically similar to the original description and are uniform in the proportionality of width on length, but exhibited different patterns of size associated with each host species. Furthermore, a genotypic difference of 3% was found in the COI sequences from T. flavipes and T. albicollis. Therefore, an ecological discussion is introduced aimed at associating these morphometric and genotypic differences with an ongoing speciation process.

Isospora borbai n. sp. (Chromista: Apicomplexa: Eimeriidae) from gnateaters Conopophaga spp. (Passeriformes: Tyranni: Conopophagidae) in South America.

BACKGROUND: The gnateaters Conopophaga spp. are insectivorous passerines commonly observed in high and humid forests, where they remain lodged in thin branches and, sometimes, they fly to the ground to catch insects. The insectivorous feeding habit is related to low prevalence and density of coccidians in passerines; however, several coccidian species are recorded for families of insectivorous passerines. PURPOSE: This study aimed to examine the feces from gnateaters Conopophaga spp. captured in the municipality of Barra Mansa and in the Itatiaia National Park, State of Rio de Janeiro, Southeastern Brazil, to determine what coccidian parasites were present. METHODS: Nine gnateaters were captured with mist nets. Coccidian oocysts were recovered from the fecal samples by flotation in Sheather's saturated solution. Morphological observations, line drawings, photomicrographs and measurements were made in optical microscopy and digitally edited. The molecular analysis included the study of the sequence of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, with phylogenetic reconstructions based on the neighbor-joining and maximum likelihood analysis. RESULTS: Four Conopophaga spp. were positive for oocysts. An Isospora sp. considered as new to science is described and identified from Conopophaga melanops (Vieillot, 1818) and Conopophaga lineata (Wied, 1831). Isospora borbai n. sp. has oocysts that are subspheroidal, 17-22 × 15-22 (20.2 × 19.1) µm, with rough, bilayered wall, c.1.7 µm thick. Micropyle present, but without micropyle cap. Oocyst residuum absent, but one or two polar granules are present. Sporocysts are ellipsoidal, 12-15 × 8-11 (14.1 × 9.1) µm. The Stieda body is knob-like to half-moon-shaped and sub-Stieda body is rounded. Sporocyst residuum is present, composed of scattered spherules of different sizes. Sporozoites are vermiform with refractile body and nucleus. Molecular analysis at the cox1 gene exhibited similarity greater than 99% with Isospora spp. isolates from other Neotropical passerine birds. CONCLUSION: Based on the morphological and molecular features, I. borbai is considered as new to science and the first coccidian species recorded from Conopophagidae.

The vulnerable Sporophila frontalis (Verreaux) and Haplospiza unicolor Cabanis as new hosts for Isospora sporophilae Carvalho-Filho, Meireles, Ribeiro & Lopes, 2005 (Eimeriidae) in Brazil.

Isospora sporophilae Carvalho-Filho, Meireles, Ribeiro & Lopes, 2005 was morphologically and molecularly identified from the double-collared seedeater Sporophila frontalis (Verreaux), which is categorised as 'vulnerable' by the International Union for Conservation of Nature and Natural Resources (IUCN), and from the uniform finch Haplospiza unicolor Cabanis in conserved and anthropomorphic/fragmented areas of Atlantic Forest in the southeastern Brazil. The oöcysts recovered from S. frontalis and H. unicolor had small morphological and genotypic differences that were not considered sufficient for the description of new species, but only different genotypes of I. sporophilae related to each host. This coccidian species was originally described from double-collared seedeaters Sporophila caerulescens (Vieillot) in a center screening of wild animals; therefore, this new report emphasises a potential occurrence of anthropomorphic dispersion of coccidia through illegal trade, seizures and reintroductions in the wild.

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.

Typical Brazilian genotype of Toxoplasma gondii isolated from a horse destined for human consumption in Europe from a slaughterhouse.

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. Infections occur via the ingestion of oocysts, consumption of cysts containing bradyzoites, and transplacental transmission of tachyzoites. Diversity in T. gondii strains may affect the outcome of clinical toxoplasmosis. The consumption of horse meat is a common practice in some parts of the world. The objectives of the present study were to isolate and genotype T. gondii from horses from an abattoir in the state of Rio Grande do Sul in southern Brazil that exports horse meat to Europe. Antibodies to T. gondii were found in 32.5% (13/40) of the horses using the modified agglutination test (MAT) with a cut-off of 1:25. Tissues from the 13 seropositive horses were bioassayed in mice, and one isolate, designated TgHorseBrRS1, was obtained. PCR-RFLP of the isolate revealed the ToxoDB-RFLP #228 genotype, a typical non-archetypal Brazilian genotype, and microsatellite analysis showed a unique non-archetypal genotype. This study showed that horses from Brazil can harbor viable T. gondii in their tissues, suggesting that recommendations to consumers should be made, especially in European countries where consumption of raw horse meat is common.

Occurrence and molecular characterization of Giardia duodenalis cysts and Cryptosporidium oocysts in raw water samples from the Rímac River, Peru.

Giardia and Cryptosporidium are potentially pathogenic protozoa which are ubiquitous in ambient surface water. The present study included 60 samples of surface water from three sampling sites from the Rímac River, Lima and Callao, Peru, to detect the occurrence of Giardia spp. and Cryptosporidium spp. and to perform molecular characterization of specimens found. Water samples were concentrated using the membrane filtration technique, and following elution, cysts and oocysts were visualized by direct immunofluorescence assay (IFA). For molecular characterization, tpi and bg gene fragments and 18S rRNA were amplified by nested PCR for Giardia and Cryptosporidium, respectively, followed by sequencing and phylogenetic analysis. Giardia cysts were found in 93.3% of the analyzed samples, whereas Cryptosporidium oocysts were detected in 15%. The positivity of the Giardia cysts was 86.6% (n = 26) in 2014, while Cryptosporidium oocysts were not detected. In 2015, both protozoa were found in raw water samples, with all 30 samples collected positive for Giardia cysts (100.0%) and 9 positive for Cryptosporidium oocysts (30.0%). Oocysts were detected in 20.0% of water samples from sites 1 (mean 5.25 oocysts/L) and 2 (mean 52.3 oocysts/L), while at site 3, oocysts were detected in 50.0% of raw water samples (mean 193.6 oocysts/L). The presence of Giardia duodenalis assemblage A was confirmed in several samples by the phylogenetic positioning of the bg and tpi genes, and the sub-assemblage AII was predominant (8/9). Sequencing for Cryptosporidium resulted in profiles compatible with Cryptosporidium hominis, Cryptosporidium meleagridis, and Cryptosporidium baileyi. This is the first time that the presence of G. duodenalis assemblage A/sub-assemblage AII and Cryptosporidium species has been reported in surface water samples in Peru. These Cryptosporidium species and the Giardia duodenalis assemblage are associated with human disease which highlights the potential risk to public health and the need to increase environmental monitoring measures to protect this water body.

Genetic uniqueness of Cryptosporidium parvum from dairy calves in Colombia.

Fecal specimens from 432 pre-weaned calves younger than 35 days were collected over a 2-year period (2010-2012) from 74 dairy cattle farms in the central area of Colombia. These samples were microscopically examined for the presence of Cryptosporidium oocysts, and positive specimens were selected for molecular examination. Microscopy revealed that 115 calves (26.6%) from 44 farms (59.5%) tested positive. Oocyst shedding was recorded in calves aged 3-day-old onwards, although the infection rate peaked at 8-14 days (40.7%). Infection rates were higher in diarrheic (52.2%) than in non-diarrheic calves (19.9%) (p < 0.0001, χ2), and infected calves had up to seven times more probability of having diarrhea than non-infected calves. Cryptosporidium species and subtypes were successfully identified in 73 samples from 32 farms. Restriction and sequence analyses of the SSU rRNA gene revealed C. parvum in all but two isolates identified as Cryptosporidium bovis. Sequence analyses of the 60-KDa glycoprotein (gp60) gene revealed eight subtypes within the IIa family. An unusual subtype (IIaA18G5R1) was the most prevalent and widely distributed (more than 66% specimens and 68% farms) while the subtype most frequently reported in cattle worldwide (IIaA15G2R1) was found in less than 13% of specimens and 16% farms. The remaining subtypes (IIaA16G2R1, IIaA17G4R1, IIaA20G5R1, IIaA19G6R1, IIaA20G6R1, and IIaA20G7R1) were restricted to 1-3 farms. This is the first large-sample size study of Cryptosporidium species and subtypes in Colombia and demonstrates the genetic uniqueness of this protozoan in cattle farms in this geographical area.

Isolation and biological and molecular characterization of Neospora caninum (NC-SP1) from a naturally infected adult asymptomatic cattle (Bos taurus) in the state of São Paulo, Brazil.

The biological and genetic diversity of Neospora caninum is very limited because of availability of only a few viable isolates worldwide. This study describes the isolation and biological and molecular characterization of a new viable isolate of N. caninum (NC-SP1), from a cattle in Brazil. Approximately 400 g of brain from a naturally infected adult male cattle from an abattoir was fed to a 2-month-old dog. Neospora-like oocysts were observed on day 7 post-inoculation (PI) and the duration of oocyst shedding was 14 days. The DNA obtained from oocysts was characterized molecularly and the final sequence was 99% identical to homologous sequences of N. caninum available in GenBank®. For bioassay, gerbils (Meriones unguiculatus) were orally inoculated with 10 100 and 1000 oocysts; all gerbils remained clinically normal but developed N. caninum antibodies 14 days PI. Cell culture isolation was successful using the brain homogenate from one of the gerbils and tachyzoites were observed 24 days PI. Microsatellite genotyping revealed a unique genetic profile for this new reference isolate.