An overview of the trypanosomatid (Kinetoplastida: Trypanosomatidae) parasites infecting several mammal species in Colombia.

BACKGROUND: Trypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution. METHODS: We used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses. RESULTS: One hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia. CONCLUSIONS: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.

Blood Feeding Sources of Nyssomyia antunesi (Diptera: Psychodidae): A Suspected Vector of Leishmania (Kinetoplastida: Trypanosomatidae) in the Brazilian Amazon.

Present work aimed to identify blood feeding sources and attempt to detect Leishmania DNA in Nyssomyia antunesi, suspected vector of Leishmania sp., from a park in the urban center of Belém, the capital of Pará State, in the Brazilian Amazon. Entire bodies and gut contents of Ny. antunesi engorged females, previously captured in the urban park with Centers for Disease Control (CDC) light traps and aspiration on tree bases, were subjected to Leishmania and vertebrate DNA detection through amplification of the Leishmania mini-exon and vertebrate cytochrome b (cyt b) gene regions, respectively. The quality of DNA extraction from entire bodies was ensured through amplification of the dipteran cyt b region. The vertebrate cyt b amplicons were sequenced and compared with those available on GenBank. A maximum likelihood phylogenetic tree was constructed to assess the clustering patterns of these sequences. Leishmania DNA was not detected. The sequences of 13 vertebrate cyt b amplicons were considered informative, exhibiting similarity and clustering with the following six vertebrate species: Dasyprocta leporina (1), Cuniculus paca (1), Tamandua tetradactyla (4), Choloepus didactylus (4), Pteroglossus aracari aracari (2), Homo sapiens (1). The samples of D. leporina and C. paca were obtained from the CDC canopy, whereas the others were by aspiration from tree bases. The present results revealed the eclectic and opportunist blood-feeding behavior of Ny. antunesi, with birds and mammals, these last ones acting as potential reservoirs for Leishmania species, distributed throughout the vertical forest strata.

Infection susceptibility and vector competence of Rhodnius robustus Larrousse, 1927 and R. pictipes Stal, 1872 (Hemiptera, Reduviidae, Triatominae) for strains of Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) I, II and IV.

BACKGROUND: Rhodnius robustus and Rhodnius pictipes are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease (CD), that are found in the Brazilian Amazon region. Susceptibility to infection and vector competence depend on the parasite-vector relationship. Our objective was to evaluate the interaction between T. cruzi and these two triatomine vectors in pure and mixed experimental infections of T. cruzi strains from the same or different geographic regions. METHODS: Fifth-instar nymphs of R. robustus and R. pictipes were fed on mice infected with four T. cruzi strains, namely genotypes TcIAM, TcIMG, TcIIPR, and TcIVAM, respectively, from the Brazilian states of Amazonas, Minas Gerais and Paraná. Over a period of 120 days, excreta were examined every 20 days to assess vector competence, and intestinal contents (IC) were examined every 30 days to determine susceptibility to infection. RESULTS: The highest positive rate in the fresh examination (%+FE, 30.0%), the highest number of parasitic forms (PF, n = 1969) and the highest metacyclogenesis rate (%MC, 53.8%) in the excreta were recorded for R. robustus/TcIVAM. Examination of the IC of R. pictipes revealed a higher number of PF in infections with TcIAM (22,680 PF) and TcIIPR (19,845 PF) alone or in association (17,145 PF), as well as a %+FE of 75.0% with TcII, in comparison with the other genotypes. The highest %MC (100%) was recorded for the mixed infections of TcIAM with TcIIPR or TcIVAM in the IC of R. pictipes. CONCLUSIONS: Overall, both species were found to be susceptible to the T. cruzi strains studied. Rhodnius robustus showed vector competence for genotypes TcIVAM and TcIAM+TcIVAM and R. pictipes for TcIAM+TcIVAM and TcIAM+TcIIPR; there was elimination of infective forms as early as at 20 days. Our results suggest that both the genetics of the parasite and its geographic origin influence the susceptibility to infection and vector competence, alone or in association.

Revisiting the cave-dwelling sand flies (Diptera, Psychodidae, Phlebotominae) from Brazil: Diversity and potential role in the transmission of Leishmania Ross, 1903 (Kinetoplastida: Trypanosomatidae).

Brazilian caves, one of the many tourist attractions of the country, may act as a shelter for insects, such as sand flies (Diptera: Psychodidae), natural hosts of various microorganisms including parasites of the genus Leishmania Ross, 1903. In the last decades, with the increasing global need for sustainable development, ecotourism has emerged as one of the major activities in Brazil. However, the constant monitoring in environmentally protected areas is not often carried out, endangering visitors and professionals, especially due to the occurrence of zoonoses. Several sand fly species have already been recorded in Brazilian caves, drawing attention to the possibility of Leishmania transmission at this ecotope. Indeed, this current systematic review summarizes the fauna of cave-dwelling sand flies in Brazil, focusing on their biological behaviour and the occurrence of potential vectors of Leishmania parasites.

Infections by trypanosomatid (Kinetoplastida: Trypanosomatidae) in triatomines (Hemiptera: Triatominae): A spatiotemporal assessment in an endemic area for Chagas disease.

This research analysed the spatiotemporal distribution of triatomines infected by trypanosomatid parasites in an endemic region for Chagas disease, in the state of Pernambuco, Northeastern Brazil. The database included the total number of triatomines captured from intradomicile and peridomicile areas, as well as the infection rate (IR) by trypanosomatid. The Gi∗ by Getis-Ord method was used to statistically identify significant concentration clusters and the IR of triatomines by trypanosomatids. A generalized linear regression model with a binomial distribution was used to evaluate the probability of finding an IR by trypanosomatids. Overall, of 4,800 triatomines examined, trypanosomatid forms similar to Trypanosoma cruzi were detected in 10.29% of them, and the majority of positive specimens (98.17%) were collected at intradomicile. The geospatial analyses identified triatomines clusters in intradomicile and peridomicile environments. According to the logistic regression data for species (Panstrongylus lutzi, P. megistus, Triatoma brasiliensis and T. pseudomaculata), the probability of detection of T. cruzi infection remains constant in up to 50 specimens examined or more. The findings of this research revealed a scenario never studied in this area through this type of spatiotemporal analysis, which is essential to identify areas of vulnerability for the occurrence of these vectors and consequently for Chagas disease.

Distribution of Merlin in eukaryotes and first report of DNA transposons in kinetoplastid protists.

DNA transposons are defined as repeated DNA sequences that can move within the host genome through the action of transposases. The transposon superfamily Merlin was originally found mainly in animal genomes. Here, we describe a global distribution of the Merlin in animals, fungi, plants and protists, reporting for the first time their presence in Rhodophyceae, Metamonada, Discoba and Alveolata. We identified a great variety of potentially active Merlin families, some containing highly imperfect terminal inverted repeats and internal tandem repeats. Merlin-related sequences with no evidence of mobilization capacity were also observed and may be products of domestication. The evolutionary trees support that Merlin is likely an ancient superfamily, with early events of diversification and secondary losses, although repeated re-invasions probably occurred in some groups, which would explain its diversity and discontinuous distribution. We cannot rule out the possibility that the Merlin superfamily is the product of multiple horizontal transfers of related prokaryotic insertion sequences. Moreover, this is the first account of a DNA transposon in kinetoplastid flagellates, with conserved Merlin transposase identified in Bodo saltans and Perkinsela sp., whereas it is absent in trypanosomatids. Based on the level of conservation of the transposase and overlaps of putative open reading frames with Merlin, we propose that in protists it may serve as a raw material for gene emergence.

Molecular Detection of Leishmania (L.) mexicana (Kinetoplastida: Trypanostomatidae) DNA in Culicoides furens (Diptera: Ceratopogonidae) from an Area with Autochthonous Canine Leishmaniasis in Northwestern Mexico.

PURPOSE: Mexico is considered endemic for Leishmania; recent reports indicate autochthonous human and canine leishmaniasis caused by Leishmania mexicana in Sinaloa state. Lutzomyia sand fly are the primary vector of the parasite, although no records of phlebotomine vectors of Leishmania exist from Sinaloa. Other hematophagous dipterans, like Culicoides, could represent possible vectors of Leishmania in absence of phlebotomines. The known distribution of Culicoides includes the southern portion of Sinaloa state, in northwestern Mexico, with records of Culicoides furens. However, no studies have demonstrated the presence of Leishmania in C. furens or its possible participation in the parasite's life cycle in Mexico. This study, therefore, sought to detect DNA of Leishmania in C. furens captured in an endemic area of autochthonous canine leishmaniasis in northwestern Mexico. METHODS: Culicoides were captured with CDC light traps, identified morphologically, and organized in pools. DNA was extracted, and used to amplify the ribosomal ITS1 region of Leishmania. PCR products were digested with HaeIII endonuclease; the banding patterns obtained were compared to reference strains. RESULTS: Leishmania mexicana DNA was detected in five out of nine pools (55%) of female C. furens. CONCLUSION: This study offers the first evidence of L. mexicana DNA in C. furens, in an endemic area of canine leishmaniasis in northwestern Mexico, where no evidence exists of the presence of phlebotomine sand fly.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Cryptobia iubilans Infections in Discus Fish in Trinidad and Tobago.

Discus (Symphysodon spp.) are costly and prized specimens in the international ornamental fish trade. The majority of discus submitted to the Aquatic Animal Health Unit at the University of the West Indies School of Veterinary Medicine for necropsy between September 2010 and September 2015 had lesions consistent with Cryptobia iubilans infection, thus prompting this study. To determine the prevalence of the flagellated gastrointestinal protozoan C. iubilans in discus fish, 32 discus were sourced from 10 suppliers, including breeders, importers, and hobbyists across Trinidad. Fish were euthanized, and the internal organs, particularly the stomach and intestine, were observed under a light microscope for characteristic granulomatous lesions and/or live C. iubilans parasites. All wet-mount slides on which granulomas were observed were also Ziehl-Neelsen acid-fast stained to presumptively exclude the presence of Mycobacterium spp., the main differential when diagnosing C. iubilans-associated granulomatous gastritis or to determine the presence of dual infections. Further histological analyses were performed on stomach and intestinal sections, and transmission electron microscopy was used to confirm the parasite in stomach sections. The prevalence of C. iubilans infection was found to be 81.3%, and the prevalence of presumptive dual infections with Mycobacterium spp. was found to be 21.9%. To the best of our knowledge, this is the first documented study of C. iubilans infections in the wider Caribbean region.

Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids.

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

Trypanosomatids (Protozoa: Kinetoplastida) in three species of Armored Catfish from Mogi-Guaçu river, Pirassununga, São Paulo, Brazil.

Trypanosome infections have been reported in several species of fish, in majority of cases described on the basis of morphological characteristics. Trypanosomes in fish are heteroxenous and transmitted by hirudineans. This study aims to evaluate the prevalence and density of infections by Trypanosoma sp. in blood from three species of catfish, Hypostomus regani, H. strigaticeps, H. albopunctatus, from the Mogi Guaçu River, Pirassununga, São Paulo, Brazil. Further, this study intends to characterize the Trypanosoma specimens found in the blood of these fish by morphological and molecular techniques. The trypanosomes overall prevalence observed was 47.6% with a general average density of 0.75 parasites/µl of blood. Hypostomus regani and Hypostomus strigaticeps showed a significant difference in prevalence. The average densities of parasites were not significantly different among the three fish species. Similar findings were observed for the monthly variations in densities. The parasites found in the three species of catfish studied showed similar morphological characteristics. The morphological data and the statistical analyses used in this study didn't show the formation of groups. The analyses provided evidence of the presence of pleomorphisms in the trypanosomes found in the three studied fish.

Trypanosomatids (Protozoa: Kinetoplastida) in three species of Armored Catfish from Mogi-Guaçu river, Pirassununga, São Paulo, Brazil / Tripanossomatídeos (Protozoa: Kinetoplastida) em três espécies de cascudos do rio Mogi-Guaçu, Pirassununga, São Paulo, Brasil

Trypanosome infections have been reported in several species of fish, in majority of cases described on the basis of morphological characteristics. Trypanosomes in fish are heteroxenous and transmitted by hirudineans. This study aims to evaluate the prevalence and density of infections by Trypanosoma sp. in blood from three species of catfish, Hypostomus regani, H. strigaticeps, H. albopunctatus, from the Mogi Guaçu River, Pirassununga, São Paulo, Brazil. Further, this study intends to characterize the Trypanosoma specimens found in the blood of these fish by morphological and molecular techniques. The trypanosomes overall prevalence observed was 47.6% with a general average density of 0.75 parasites/µl of blood. Hypostomus regani and Hypostomus strigaticeps showed a significant difference in prevalence. The average densities of parasites were not significantly different among the three fish species. Similar findings were observed for the monthly variations in densities. The parasites found in the three species of catfish studied showed similar morphological characteristics. The morphological data and the statistical analyses used in this study didnt show the formation of groups. The analyses provided evidence of the presence of pleomorphisms in the trypanosomes found in the three studied fish.(AU)

Infecções por tripanossomas foram descritas em diversas espécies de peixes, sendo a maioria das descrições baseada nas características morfológicas. Tripanossomas de peixes são heteroxenos e transmitidos por hirudíneos. Este estudo tem como objetivo a avaliação da prevalência e densidade da infecção por Trypanosoma sp. no sangue de três espécies de cascudos, Hypostomus regani, H.strigaticeps, H.albopunctatus, do Rio Mogi-Guaçu, Pirassununga, São Paulo, Brasil. Além disso, pretende-se a caracterização das espécies de Trypanosoma encontradas no sangue desses peixes, por meio da utilização de técnicas morfólogicas e moleculares. A prevalência total de tripanossomas foi de 47,6%, e a densidade média foi de 0,75 parasitas/ µl de sangue. Hypostomus regani e Hypostomus strigaticeps apresentaram diferenças significativas em prevalência. As médias das densidades dos parasitas não apresentaram diferenças significativas entre as três espécies de peixes estudadas. O mesmo foi observado com a variação mensal das densidades. Os parasitas encontrados nas três espécies de cascudo possuíam características morfológicas semelhantes. As análises morfológicas e estatísticas obtidas neste estudo não mostraram a formação de grupos. As análises evidenciaram a presença de pleomorfismo dos tripanossomas encontrados nas três espécies de peixes estudados.(AU)

Trypanosomatids (Protozoa: Kinetoplastida) in three species of Armored Catfish from Mogi-Guaçu river, Pirassununga, São Paulo, Brazil / Tripanossomatídeos (Protozoa: Kinetoplastida) em três espécies de cascudos do rio Mogi-Guaçu, Pirassununga, São Paulo, Brasil

Abstract Trypanosome infections have been reported in several species of fish, in majority of cases described on the basis of morphological characteristics. Trypanosomes in fish are heteroxenous and transmitted by hirudineans. This study aims to evaluate the prevalence and density of infections by Trypanosoma sp. in blood from three species of catfish, Hypostomus regani, H. strigaticeps, H. albopunctatus, from the Mogi Guaçu River, Pirassununga, São Paulo, Brazil. Further, this study intends to characterize the Trypanosoma specimens found in the blood of these fish by morphological and molecular techniques. The trypanosomes overall prevalence observed was 47.6% with a general average density of 0.75 parasites/µl of blood. Hypostomus regani and Hypostomus strigaticeps showed a significant difference in prevalence. The average densities of parasites were not significantly different among the three fish species. Similar findings were observed for the monthly variations in densities. The parasites found in the three species of catfish studied showed similar morphological characteristics. The morphological data and the statistical analyses used in this study didn’t show the formation of groups. The analyses provided evidence of the presence of pleomorphisms in the trypanosomes found in the three studied fish.

Resumo Infecções por tripanossomas foram descritas em diversas espécies de peixes, sendo a maioria das descrições baseada nas características morfológicas. Tripanossomas de peixes são heteroxenos e transmitidos por hirudíneos. Este estudo tem como objetivo a avaliação da prevalência e densidade da infecção por Trypanosoma sp. no sangue de três espécies de cascudos, Hypostomus regani, H.strigaticeps, H.albopunctatus, do Rio Mogi-Guaçu, Pirassununga, São Paulo, Brasil. Além disso, pretende-se a caracterização das espécies de Trypanosoma encontradas no sangue desses peixes, por meio da utilização de técnicas morfólogicas e moleculares. A prevalência total de tripanossomas foi de 47,6%, e a densidade média foi de 0,75 parasitas/ µl de sangue. Hypostomus regani e Hypostomus strigaticeps apresentaram diferenças significativas em prevalência. As médias das densidades dos parasitas não apresentaram diferenças significativas entre as três espécies de peixes estudadas. O mesmo foi observado com a variação mensal das densidades. Os parasitas encontrados nas três espécies de cascudo possuíam características morfológicas semelhantes. As análises morfológicas e estatísticas obtidas neste estudo não mostraram a formação de grupos. As análises evidenciaram a presença de pleomorfismo dos tripanossomas encontrados nas três espécies de peixes estudados.

Selenoproteins of African trypanosomes are dispensable for parasite survival in a mammalian host.

The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era.

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Diagnóstico paleoparasitológico de protozoários do gêneroLeishmania Ross, 1903 (Kinetoplastida, Trypanosomatidae)em material arqueológico da América do Sul / Parasitological diagnosis paleo protozoa of the genus Leishmania Ross, 1903 (Kinetoplastida, Trypanosomatidae) in archaeological material from South America

Espécies do gênero Leishmania (Kinetoplastida, Trypanosomatidae) são agentescausadores de leishmanioses, que representam um complexo de doenças com variado espectroclínico e diversidade epidemiológica que, em alguns países, constituem-se em grave problema de saúde pública. A origem e evolução das leishmanioses têm estado em debate em seus aspectos clínicos e parasitológicos. Com a introdução da paleoparasitologia baseada na aplicação de diferentes técnicas, tornou-se possível a recuperação de vestígios do parasito,assim como o diagnóstico de infecções em humanos e em outros hospedeiros no passado. Os objetivos desta pesquisa foram diagnosticar parasitos do gênero Leishmania spp., em materiais arqueológicos da América do Sul por meio de técnicas moleculares e imunológicas. Estes são os primeiros registros de recuperação de DNA de Leishmania sp. em material arqueológico do Brasil, assim como também o primeiro registro deL. tarentolae em material antigo. Dois pares de primers que amplificam outras regiões de L. tarentolae foram desenhados para o estudo no intuito de averiguar se outros trechos de DNA desta espécie também teriam se preservado no material, tendo os mesmos não amplificado.Duas explicações são possíveis: devido a natureza fragmentada do DNA antigo o mesmo não se conservou de maneira homogenia no material, ou pode se tratar de um infectron, o que também é um dado relevante pois é uma assinatura da relação estabelecida entre parasito e hospedeiro...

Species of the genus Leishmania (Kinetoplastida, Trypanosomatidae) are causativeagents of leishmaniasis, which represents a complex disease with varied clinical spectrum and epidemiological diversity that, in some countries, constitute a serious public health problem. The origin and evolution of leishmaniasis has been under discussion in clinical and parasitological aspects. By the introduction of paleoparasitology based on the application of different techniques, as well as the diagnosis of infections in humans and other hosts in the past. Theaims of this research were: diagnosis by molecular PCR technique parasites of the genus Leishmaniain archaeological materials of South America, with the comparison of the sequences obtained from the GenBank for both recent and archaeological material, diagnosis through serological techniques: rapid test DPP and Western Blot, anti-Leishmania antibodies in the samples submitted to PCR, with subsequent comparison of these serological techniques with the previous lyused molecular technique and thus contribute to the knowledge of paleoparasitology of leishmaniasis, giving contributing to studies on the origin and evolution of Leishmania genus...

Comparative analyses of the ß-tubulin gene and molecular modeling reveal molecular insight into the colchicine resistance in kinetoplastids organisms.

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the ß-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania ß-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the ß-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α-helix structure and displacement to the inside of the pocket of one ß-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.

Whole-cell antigenicity data support sequence-based kinetoplastid taxonomy.

Early immunological data, obtained by immunodiffusion and immunoelectrophoresis, on the whole-cell antigenicity of kinetoplastid protozoa were retrieved and used to construct a dendrogram of antigenic distances. Remarkably, they supported the same taxonomic conclusions as analyses based on DNA and protein sequence data.