Phylogenetic position of the freshwater fish trypanosome, Trypanosoma ophiocephali (Kinetoplastida) inferred from the complete small subunit ribosomal RNA gene sequence

The complete small subunit rRNA (SSrRNA) gene sequence (2,142 nucleotides) of the freshwater fish trypanosome Trypanosoma ophiocephali Chen (1964) was determined. The phylogenetic analysis deduced using neighbor-joining, maximum parsimony, and Bayesian methods demonstrated the existence of an “aquatic clade”. T. ophiocephali was revealed to be a member of the freshwater fish trypanosomes and form the sister species with Trypanosoma siniperca and Trypanosoma sp. Carpio with high bootstrap values (98% MP, 100% NJ, 100% Bay). The high similarity of SSrRNA gene sequences and morphometric characters showed that T. ophiocephali, T. siniperca and T. sp. Carpio probably were the same species. The phylogenetic trees further suggested that Chinese freshwater fish trypanosome might be paraphyletic, and fish trypanosomes should have low host specificity

Infection Dynamics and Immune Response in a Newly Described Drosophila-Trypanosomatid Association

Trypanosomatid parasites are significant causes of human disease and are ubiquitous in insects. Despite the importance of Drosophila melanogaster as a model of infection and immunity and a long awareness that trypanosomatid infection is common in the genus, no trypanosomatid parasites naturally infecting Drosophila have been characterized. Here, we establish a new model of trypanosomatid infection in Drosophila-Jaenimonas drosophilae, gen. et sp. nov. As far as we are aware, this is the first Drosophila-parasitic trypanosomatid to be cultured and characterized. Through experimental infections, we find that Drosophila falleni, the natural host, is highly susceptible to infection, leading to a substantial decrease in host fecundity. J. drosophilae has a broad host range, readily infecting a number of Drosophila species, including D. melanogaster, with oral infection of D. melanogaster larvae resulting in the induction of numerous immune genes. When injected into adult hemolymph, J. drosophilae kills D. melanogaster, although interestingly, neither the Imd nor the Toll pathway is induced and Imd mutants do not show increased susceptibility to infection. In contrast, mutants deficient in drosocrystallin, a major component of the peritrophic matrix, are more severely infected during oral infection, suggesting that the peritrophic matrix plays an important role in mediating trypanosomatid infection in Drosophila. This work demonstrates that the J. drosophilae-Drosophila system can be a powerful model to uncover the effects of trypanosomatids in their insect hosts. IMPORTANCE Trypanosomatid parasites are ubiquitous in insects and are significant causes of disease when vectored to humans by blood-feeding insects. In recent decades, Drosophila has emerged as the predominant insect model of infection and immunity and is also known to be infected by trypanosomatids at high rates in the wild. Despite this, there has been almost no work on their trypanosomatid parasites, in part because Drosophila-specific trypanosomatids have been resistant to culturing. Here, we present the first isolation and detailed characterization of a trypanosomatid from Drosophila, finding that it represents a new genus and species, Jaenimonas drosophilae. Using this parasite, we conducted a series of experiments that revealed many of the unknown aspects of trypanosomatid infection in Drosophila, including host range, transmission biology, dynamics of infection, and host immune response. Taken together, this work establishes J. drosophilae as a powerful new opportunity to study trypanosomatid infections in insects

Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease. © 2014 Porcel et al

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Funder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health); doi: https://doi.org/10.13039/100011272; Grant(s): 305444, 305444Funder: Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness); doi: https://doi.org/10.13039/501100003329Funder: Generalitat de Catalunya (Government of Catalonia); doi: https://doi.org/10.13039/501100002809Funder: EC | European Regional Development Fund (Europski Fond za Regionalni Razvoj); doi: https://doi.org/10.13039/501100008530Funder: Instituto Nacional de Bioinformática ELIXIR Implementation Studies Centro de Excelencia Severo OchoaFunder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health)Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics

The human environment protection and development problems in the secondary school education proces in Socialist Republic of Czechoslovakia

In conformity with the suggestions of the International Union for Conservation of Nature and Natural Resources (IUCN) and the East European Education Committee (MSOP) the problems of the human environment protection and development have been introduced to all the secondary school subjects, in a variable degree, in all the member countries of the Committee. The new qualitative level of influence of the society on the nature and the natural environment of man creates a pressure on the actual range and structure of some of the school courses (biology, geography, social sciences, etc.). In the Czechoslovak secondary schools the most comprehensive and broadest approach to the environment development and protection can be accomplished within the course on society subject in the following issues: the socialist workstyle, man and his environment, natural environment versus social environment, culture and its components, countermeasures against devastation, and same others. The time assigned for biology and geography teaching is inadequately used for the education on the human natural environment. A more specialist approach to the environmental problems is offered to professional schools of such specialities as: metallurgy, (coal and ores) mining, geology, mechanical engineering, electrical engineering, chemical technology, building engineering, etc. This body of problems are considered in terms of the technical and technological needs, as well as from standpoint of legislation related to the obligations of manufacturing plants and discipline at work. Considerable emphasis is placed on the issues of the hygiene and industrial safety. The inclusion of the human environment development and protection in the educational process of Czechoslovak secondary achools results from the need of providing their graduates with comprehensive knowledge, and especially in the aspect of its practical application, the knowledge which would meet the social needs

Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala (Phascolarctos cinereus)

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0.9% at the 18S rDNA and 10.7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38.2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp

Virulent and attenuated lines of Leishmania major: DNA karyotypes and differences in metalloproteinase GP63

The Leishmania metalloproteinase GP63 has been reported to play important roles mainly in resistance of promastigotes to complement-mediated lysis and in interaction with macrophage receptors. On the other hand, its function in insect vectors is still unclear. We compared the structure and dosage of gp63 genes and the activity of GP63 in Leishmania major Yakimoff et Schokhor strains and lines differing in virulence for mice and ability to develop in sand flies. The results demonstrate considerable variability in amount and proteolytical activity of GP63 among L. major strains although genomic changes in the gp63 locus were not found. Attenuated LV561/AV line showed low amount and low enzymatic activity of GP63. Serial passages of attenuated parasites through either Phlebotomus duboscqi Neveu-Lemaire or through mice led to a recovery of GP63 proteolytical activity to the level present in virulent LV561/V line. Overexpression of GP63 was found in two L major strains (L119, Neal) with defective lipophosphoglycan (LPG); both these strains were capable to cause mice infection but unable to survive and multiply in sand flies. Differences were found also in karyotypes and in amount of minichromosomes amplified in some lines of the LV561 strain. The results suggest that parasite virulence is not simply correlated with the activity of GP63; however, this enzyme plays a significant role in association with other surface molecules, especially LPG. Overexpression of GP63 can compensate LPG defect in the vertebrate host but in sand flies both molecules fulfil quite different functions and the defect in LPG is lethal for the parasite. On the other hand, linear minichromosomes of about 200 kb found in some lines of the LV561 strain are associated with development in vitro and in the vector but they are not essential for the infection of the vertebrate host