Genetic Variation among the Partial Gene Sequences of the Ribosomal Protein Large-Two, the Internal Transcribed Spacer, and the Small Ribosomal Subunit of Blastocystis sp. from Human Fecal Samples.

In the present study, we compared the genetic variability of fragments from the internal transcribed spacer region (ITS) and the small subunit ribosomal DNA (SSUrDNA) as nuclear markers, in contrast with the ribosomal protein large two (rpl2) loci, placed in the mitochondrion-related organelles (MROs) within and among human fecal samples with Blastocystis. Samples were analyzed using polymerase chain reaction (PCR)-sequencing, phylogenies, and genetics of population structure analyses were performed. In total, 96 sequences were analyzed, i.e., 33 of SSUrDNA, 35 of rpl2, and 28 of ITS. Only three subtypes (STs) were identified, i.e., ST1 (11.4%), ST2 (28.6%), and ST3 (60%); in all cases, kappa indexes were 1, meaning a perfect agreement among ST assignations. The topologies of phylogenetic inferences were similar among them, clustering to each ST in its specific cluster; discrepancies between phylogeny and assignment of STs were not observed. The STRUCTURE v2.3.4 software assigned three subpopulations corresponding to the STs 1-3, respectively. The population indices were consistent with those previously reported by other groups. Our results suggest the potential use of the ITS and rpl2 genes as molecular markers for Blastocystis subtyping as an alternative approach for the study of the genetic diversity observed within and between human isolates of this microorganism.

Identification and validation of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening.

Blastocystis sp. is among the most frequent intestinal protists identified in humans globally. However, characterization of Blastocystis subtype diversity in humans is ongoing. We report here the identification of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening involving colonoscopy and fecal testing (microscopy, culture, PCR). The full-length ssu rRNA gene sequence of the protist was generated using MinION long-read sequencing technology. The validity of the novel subtype was confirmed via phylogenetic and pairwise distance analyses of the full-length ST41 sequence and all other valid subtypes. The study provides reference material essential for conducting subsequent experimental studies.

From Argentine Abyssal Plain to farmed turbot in Spain: A ubiquitous amoeba species Vannella robusta sp. nov. (Amoebozoa, Vannellida).

A strain with the characters of the genus Vannella was isolated from the water layer immediately above the deep-sea sediment collected in the south-western Atlantic Ocean, ca. 4.6 km deep. Small-subunit ribosomal RNA (SSU rRNA) and cytochrome c oxidase (Cox1) gene phylogenetic analyses showed that the new strain branches within the clade of previously isolated unnamed Vannella strains from different marine fish and invertebrate hosts. Although the SSU rRNA gene sequences of these strains show variability within 2% of all nucleotide positions without any regular pattern, the available Cox1 gene sequences from within this clade are identical. Given the morphological homogeneity of the revealed clade, all of its strains can be assigned under the same species name, and the variation of their SSU rRNA is comparable to its intragenomic variation, as shown by molecular cloning of the PCR amplicons. High variability of the SSU rRNA gene sequences within and between independently isolated morphologically identical strains in combination with highly conserved Cox1 gene sequences may be a feature in some clades of Vannella, but is not a general rule for this genus, as SSU rRNA genes conserved between different morphospecies occur in several other clades within Vannella.

Fecal bacterial communities of wild black capuchin monkeys (Sapajus nigritus) from the Atlantic Forest biome in Southern Brazil are divergent from those of other non-human primates.

Gut microbiota are influenced by factors such as diet, habitat, and social contact, which directly affect the host's health. Studies related to gut microbiota in non-human primates are increasing worldwide. However, little remains known about the gut bacterial composition in wild Brazilian monkeys. Therefore, we studied the fecal microbiota composition of wild black capuchin monkey (Sapajus nigritus) (n=10) populations from two different Atlantic Forest biome fragments (five individuals per fragment) in south Brazil. The bacterial community was identified via the high-throughput sequencing and partial amplification of the 16S rRNA gene (V4 region) using an Ion Personal Genome Machine (PGMTM) System. In contrast to other studies involving monkey microbiota, which have generally reported the phyla Firmicutes and Bacteroidetes as predominant, black capuchin monkeys showed a high relative abundance of Proteobacteria ( χ ¯ = 80.54%), followed by Firmicutes ( χ ¯ = 12.14%), Actinobacteria ( χ ¯ = 4.60%), and Bacteriodetes ( χ ¯ = 1.31%). This observed particularity may have been influenced by anthropogenic actions related to the wild habitat and/or diet specific to the Brazilian biome's characteristics and/or monkey foraging behavior. Comparisons of species richness (Chao1) and diversity indices (Simpson and InvSimpson) showed no significant differences between the two groups of monkeys. Interestingly, PICRUSt2 analysis revealed that metabolic pathways present in the bacterial communities were associated with xenobiotic biodegradation and the biosynthesis of secondary metabolites, which may suggest positive effects on monkey health and conservation in this anthropogenic habitat. Infectious disease-associated microorganisms were also observed in the samples. The present study provides information about the bacterial population and metabolic functions present in fecal microbiota, which may contribute to a better understanding of the ecology and biology of black capuchin monkeys living in forest fragments within the Atlantic Forest biome in southern Brazil. Additionally, the present study demonstrates that the fecal bacterial communities of wild black capuchin monkeys in this area are divergent from those of other wild non-human primates.

Abdominal dioctophymosis in a domestic cat from the Peruvian rainforest confirmed morphologically and molecularly.

A case of abdominal dioctophymosis in a domestic cat was found in San Juan Bautista district, the Peruvian rainforest, in the Loreto department of Peru. The pet went to a veterinary clinic for a routine ovariohysterectomy during which a large nematode was found in the abdominal cavity. The nematode was morphologically identified as an adult female of Dioctophyme sp. A few morphological parameters, such as the vagina distance from the anterior part and the egg size, were different than D. renale. Partial sequences of the cytochrome c oxidase subunit I (cox1) and the small subunit 18S ribosomal RNA genes were compared with the references from public sequence database and showed a genetic identifies of 89.25% and 99.65% with D. renale, respectively. This is the first mitochondrial molecular analysis of a Dioctophyme specimen from South America and the results showed up to 12.5% nucleotide sequence variation in cox 1 gene of D. renale.

Epidemiology, species composition and genetic diversity of tetra- and octonucleated Entamoeba spp. in different Brazilian biomes.

BACKGROUND: Entamoeba species harbored by humans have different degrees of pathogenicity. The present study explores the intra- and interspecific diversity, phylogenetic relationships, prevalence and distribution of tetra- and octonucleated cyst-producing Entamoeba in different Brazilian regions. METHODS: Cross-sectional studies were performed to collect fecal samples (n = 1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado, and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis. RESULTS: Light microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6-10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four different species were identified: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n = 32) were characterized; however, new ribosomal lineages were not identified. The Entamoeba coli ST1 subtype predominated in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histolytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the first containing uni- and tetranucleated and the second containing uni- and octonucleated cyst-producing Entamoeba species. Molecular diversity indexes revealed a high interspecific diversity for tetra- and octonucleated Entamoeba spp. (H ± SD = 0.9625 ± 0.0126). The intraspecific diversity varied according to species or subtype: E. dispar and E. histolytica showed lower diversity than E. coli subtypes ST1 and ST2 and E. hartmanni. CONCLUSIONS: Tetra- and octonucleated cyst-producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a field to be further explored and provides information with important implications for public health.

Prevalence and molecular characterization of Cryptosporidium spp. in Père Davids deer (Elaphurus davidianus) in Jiangsu, China / Prevalência e caracterização molecular de Cryptosporidium spp. no cervo de Père David (Elaphurus davidianus) em Jiangsu, China

Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père Davids deer. In this study, 137 fecal samples from Père Davids deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père Davids deer in this area.(AU)

Cryptosporidium é um parasita zoonótico que causa diarreia em uma ampla gama de animais, incluindo veados. Pouco se sabe sobre a prevalência e o genótipo de Cryptosporidium spp. no cervo de Père David. Neste estudo, 137 amostras fecais do cervo de Père David foram coletadas entre julho de 2017 e agosto de 2018, na Reserva Dafeng, e analisadas para Cryptosporidium spp. por nested-PCR baseado no gene do RNA ribossômico da subunidade pequena (SSU rRNA), seguido de análises de sequências para determinar as espécies. O gene da glicoproteína de 60 kDa (gp60) foi utilizado para caracterizar Cryptosporidium spp. Dentre as 137 amostras, 2 (1,46%) foram positivas para Cryptosporidium spp. de acordo com os resultados do sequenciamento gênico de SSU rRNA. Ambas as amostras pertenciam ao genótipo do cervo Cryptosporidium, com duas deleções nucleotídicas e uma substituição nucleotídica. Os dados de prevalência e a caracterização molecular deste estudo fornecem conhecimentos básicos para controlar e prevenir infecções por Cryptosporidium nos cervos de Père David nessa.(AU)

Prevalence and molecular characterization of Cryptosporidium spp. in Père Davids deer (Elaphurus davidianus) in Jiangsu, China / Prevalência e caracterização molecular de Cryptosporidium spp. no cervo de Père David (Elaphurus davidianus) em Jiangsu, China

Abstract Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David's deer. In this study, 137 fecal samples from Père David's deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David's deer in this area.

Resumo Cryptosporidium é um parasita zoonótico que causa diarreia em uma ampla gama de animais, incluindo veados. Pouco se sabe sobre a prevalência e o genótipo de Cryptosporidium spp. no cervo de Père David. Neste estudo, 137 amostras fecais do cervo de Père David foram coletadas entre julho de 2017 e agosto de 2018, na Reserva Dafeng, e analisadas para Cryptosporidium spp. por nested-PCR baseado no gene do RNA ribossômico da subunidade pequena (SSU rRNA), seguido de análises de sequências para determinar as espécies. O gene da glicoproteína de 60 kDa (gp60) foi utilizado para caracterizar Cryptosporidium spp. Dentre as 137 amostras, 2 (1,46%) foram positivas para Cryptosporidium spp. de acordo com os resultados do sequenciamento gênico de SSU rRNA. Ambas as amostras pertenciam ao genótipo do cervo Cryptosporidium, com duas deleções nucleotídicas e uma substituição nucleotídica. Os dados de prevalência e a caracterização molecular deste estudo fornecem conhecimentos básicos para controlar e prevenir infecções por Cryptosporidium nos cervos de Père David nessa.

Is Leishmania (Viannia) braziliensis parasite load associated with disease pathogenesis?

BACKGROUND: Leishmania (Viannia) braziliensis is the main etiological agent of tegumentary leishmaniasis in the Americas. Parasite molecular diversity and host immune status contribute to extensive variations in its clinical presentation within endemic areas of Brazil. Pentavalent antimonials have been used for more than 60 years as the first-line drug for all cases, despite the potential for severe side effects and refractoriness. In Rio de Janeiro, Brazil, most L. (V.) braziliensis infections are benign with a scarcity of parasites, although metastasis and refractory infections can arise. In this scenario, the use of novel molecular tools can be useful for diagnosis and to assess tissue parasitism, and is of benefit to clinical and therapeutic management. METHODS: In this study, parasite load was assessed by real-time PCR based on the leishmanial small subunit ribosomal RNA gene. RESULTS AND CONCLUSION: The data revealed a tendency to higher tissue parasitism in the skin compared to mucous lesion sites and a reduction with disease progression. Parasite load was lower in poor compared to good responders to antimonials, and was also reduced in recurrent lesions compared to primary ones. However, parasite load became higher with sequential relapses, pointing to an immune system inability to control the infection. Therefore the parasite burden does not seem to be a good predictor of disease progression.

Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon.

BACKGROUND: The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. METHODS: We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. FINDINGS: Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. INTERPRETATION: This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts.

Morphology and Phylogeny of Prorocentrum texanum sp. nov. (Dinophyceae): A New Toxic Dinoflagellate from the Gulf of Mexico Coastal Waters Exhibiting Two Distinct Morphologies.

A new planktonic species of Prorocentrum is described from the Gulf of Mexico. First observed with the Imaging FlowCytobot, Prorocentrum texanum sp. nov. was characterized using LM, SEM, and TEM along with sequencing of the SSU, LSU, and ITS ribosomal regions and the mitochondrial cob and cox1 regions. P. texanum sp. nov. is a round to oval bivalvate dinoflagellate, with a prominent anterior, serrated solid flange on periflagellar a platelet and an opposing short, flat flange on the h platelet. The periflagellar area consists of 10 platelets. Both left and right valves have shallow round depressions and two-sized valve pores. The anterior ejectosome pore pattern differs between the left and right valve in relation to the periflagellar area and margins. Ten to eleven rows of tangential ejectosome pores are present on each valve. P. texanum sp. nov. has two varieties which exhibit distinct morphotypes, one round to oval (var. texanum) and the other pointed (var. cuspidatum). P. texanum var. cuspidatum is morphologically similar to P. micans in surface markings, but is smaller, and has a serrated periflagellar flange, and is genetically distinct from P. micans. Cytologically, P. texanum has two parietal chlo-roplasts, each with a compound, interlamellar pyrenoid, trichocysts, fibrous vesicles that resemble mucocysts, pusules, V- to U-shaped posterior nucleus, golgi, and tubular mitochondria. No genetic difference was found between the two varieties in the five genes examined. Phylogenetic analysis of the SSU, LSU, and ITS ribosomal regions place P. texanum sp. nov. as a sister group to P. micans. One isolate of P. texanum var. texanum produces okadaic acid.

Phylogenetic analysis of Pneumocystis from pig lungs obtained from slaughterhouses in southern and midwestern regions of Brazil / Análise filogenética de Pneumocystis obtidos de pulmões de suínos provenientes de abatedouros da região sul e centro oeste do Brasil

The Pneumocystis genus is comprised of pathogens dwelling in the lungs of terrestrial, aerial, and aquatic mammals. Occasionally they induce severe pneumonitis, particularly in hosts with severe impairment of the immune system and progressively may fill pulmonary alveolar cavities causing respiratory failure. Molecular genetic studies revealed that Pneumocystis gene sequences present a marked divergence with the host species concerned. In the present study, the genetic diversity of Pneumocystis obtained from lungs of swines was examined by analyzing mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences. The samples were obtained from two slaughterhouses located in two Brazilian states. Phylogenetic analysis demonstrated that genetic groupings within Pneumocystis organisms were in accordance with those of the corresponding hosts and that two clusters were formed. In conclusion, these data show that there are genetically distinct porcine Pneumocystis genotypes with at least two separate clusters in Brazil.

O gênero Pneumocystis compreende patógenos que residem em pulmões de animais terrestres, aéreos e aquáticos. Pode ocasionar uma grave pneumonia, particularmente em hospedeiros com o sistema imunológico seriamente comprometido, o que ocorre por meio de uma progressiva disseminação nas cavidades alveolares, causando insuficiência respiratória. Estudos genéticos, baseados em métodos moleculares, revelaram que as sequências dos genes de Pneumocystis apresentam marcante divergência de acordo com a espécie de hospedeiro. Neste estudo, a diversidade genética das amostras obtidas a partir de pulmões de suínos, provenientes de dois abatedouros localizados em dois estados brasileiros, foi examinada por análise das sequencias dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis. O resultado confirma a tendência registrada em pesquisas com amostras de outros animais e permite concluir que existem, pelo menos, dois grupos filogenéticos distintos de Pneumocystis de suínos no Brasil.

Phylogenetic analysis of Pneumocystis from pig lungs obtained from slaughterhouses in southern and midwestern regions of Brazil / Análise filogenética de Pneumocystis obtidos de pulmões de suínos provenientes de abatedouros da região sul e centro oeste do Brasil

The Pneumocystis genus is comprised of pathogens dwelling in the lungs of terrestrial, aerial, and aquatic mammals. Occasionally they induce severe pneumonitis, particularly in hosts with severe impairment of the immune system and progressively may fill pulmonary alveolar cavities causing respiratory failure. Molecular genetic studies revealed that Pneumocystis gene sequences present a marked divergence with the host species concerned. In the present study, the genetic diversity of Pneumocystis obtained from lungs of swines was examined by analyzing mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences. The samples were obtained from two slaughterhouses located in two Brazilian states. Phylogenetic analysis demonstrated that genetic groupings within Pneumocystis organisms were in accordance with those of the corresponding hosts and that two clusters were formed. In conclusion, these data show that there are genetically distinct porcine Pneumocystis genotypes with at least two separate clusters in Brazil.(AU)

O gênero Pneumocystis compreende patógenos que residem em pulmões de animais terrestres, aéreos e aquáticos. Pode ocasionar uma grave pneumonia, particularmente em hospedeiros com o sistema imunológico seriamente comprometido, o que ocorre por meio de uma progressiva disseminação nas cavidades alveolares, causando insuficiência respiratória. Estudos genéticos, baseados em métodos moleculares, revelaram que as sequências dos genes de Pneumocystis apresentam marcante divergência de acordo com a espécie de hospedeiro. Neste estudo, a diversidade genética das amostras obtidas a partir de pulmões de suínos, provenientes de dois abatedouros localizados em dois estados brasileiros, foi examinada por análise das sequencias dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis. O resultado confirma a tendência registrada em pesquisas com amostras de outros animais e permite concluir que existem, pelo menos, dois grupos filogenéticos distintos de Pneumocystis de suínos no Brasil.(AU)