Mixed human intra- and inter-subtype infections with the parasite Blastocystis sp.

Because of their limitations, current subtyping methods likely underestimate mixed human intra- and inter-subtype infections with Blastocystis sp. leading to erroneous data in the context of epidemiological studies. We confirmed this hypothesis by the identification of several isolates belonging to three subtypes in a patient considered at high risk of mixed infection through her lifestyle in rural area and long history of travelling.

Molecular phylogeny and evolution of parabasalia with improved taxon sampling and new protein markers of actin and elongation factor-1α.

BACKGROUND: Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa-in particular determining the root-is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. PRINCIPAL FINDINGS: Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. CONCLUSIONS/SIGNIFICANCE: We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids.

Molecular identification and phylogenetic relationships of trichomonad isolates of galliform birds inferred from nuclear small subunit rRNA gene sequences.

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.

Molecular characterization of a new Tetratrichomonas species in a patient with empyema.

A new Tetratrichomonas species was identified by molecular and phylogenetic approaches in the pleural fluid from a patient with encysted empyema leading to dyspnea. This observation raised the questions of the real prevalence of pulmonary trichomonosis in humans, the zoonotic potential of trichomonads, and the existence of human-host-adapted strains.

Molecular phylogeny of parabasalids with emphasis on the order Cristamonadida and its complex morphological evolution.

Parabasalia represents a complex assemblage of species, which recently received extensive reorganization. The newly created order Cristamonadida unites complex hypermastigids belonging to the Lophomonadida like the joeniids, the multinucleate polymonad Calonymphidae, and well-developed trichomonads in the Devescovinidae. All these protists exclusively occur in the guts of termites and related insects. In this study, small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase genes were identified without cultivation from 14 species in Cristamonadida including previously unstudied genera such as Joenina, Joenia, Joenoides, Macrotrichomonas, Gigantomonas, and Foaina. Despite the great morphological diversity of Cristamonadida, our phylogenetic analyses supported the monophyly of this order. However, almost all the families and subfamilies composing this order are polyphyletic suggesting a complicated morphological evolution. Our analyses also showed that Cristamonadida descends from one lineage of rudimentary trichomonads and that joeniids was basal in this order. Several successive and independent morphological transitions such as the development and reduction of flagellar apparatus and associated cytoskeleton and transition to multinucleated status have likely led to the diversity and complexity of cristamonad lineages.

Molecular characterization of iron-containing superoxide dismutases in the heterotrophic dinoflagellate Crypthecodinium cohnii.

Superoxide dismutases (SODs) are a family of antioxidant enzymes that catalyse the degradation of toxic superoxide radicals in obligate and facultative aerobic organisms. Here, we report the presence of a multi-copy gene family encoding SODs in the heterotrophic dinoflagellate Crypthecodinium cohnii. All the genes identified (sod1 to sod17) have been cloned and sequenced, and shown to encode potentially functional dimeric iron-containing SOD isozymes. Our data revealed a considerable molecular heterogeneity of this enzyme in C. cohnii at both genomic and transcriptional levels. The C. cohnii SOD1, overexpressed in Escherichia coli, was active and its structure obtained by homology modeling using X-ray crystal structures of homologues exhibited the typical fold of dimeric FeSODs. Phylogenetic studies including 110 other dimeric FeSODs and closely related cambialistic dimeric SOD sequences showed that the C. cohnii SODs form a monophyletic group and have all been acquired by the same event of horizontal gene transfer. It also revealed a dichotomy within the C. cohnii SOD sequences that could be explained by an ancestral sod gene duplication followed by subsequent gene duplications within each of the two groups. Enzyme assays of SOD activity indicated the presence of two FeSOD activities in C. cohnii cell lysate whereas MnSOD and Cu/ZnSOD were not detected. These activities contrasted with the SOD repertoire previously characterized in photosynthetic dinoflagellates. To explain these differences, a hypothetical evolutionary scenario is proposed that suggests gains and losses of sod genes in dinoflagellates.

Morphological and molecular identification of non-Tritrichomonas foetus trichomonad protozoa from the bovine preputial cavity.

Tritrichomonas foetus is the causative agent of bovine trichomonosis. This protozoan is found in the preputial cavity of bulls and is transmitted to cows during coitus. Currently, the diagnosis of this parasite is based on microscopic examination of preputial washings or scrapings, but it was recently recognized that other trichomonads similar in size, shape, and motility to T. foetus can be present in preputial samples. Despite the serious consequences of an incorrect diagnosis for bovine trichomonosis, the precise speciation of these other trichomonads has remained uncertain. Here, a total of 12 non-T. foetus isolates were microscopically examined. On the basis of morphological criteria, seven of these isolates were identified as Tetratrichomonas sp., whereas four other isolates coincided with the description of Pentatrichomonas hominis. In the last isolate, a third non-T. foetus species was identified as belonging to the genera Pseudotrichomonas or Monocercomonas: the first time that species of either of these genera have been reported in preputial samples. To confirm these data, small subunit rRNA gene sequences were obtained by PCR from the 12 trichomonad isolates. These new sequences were analysed in a broad phylogeny including 72 other parabasalid sequences. From our phylogenetic trees, we confirmed the taxonomic status of non-T. foetus organisms isolated from preputial samples (Tetratrichomonas, Pentatrichomonas, and Pseudotrichomonas) and suggested the existence of two Tetratrichomonas species, despite their morphological similarity. The route of transmission of the non-T. foetus organisms identified in the bovine preputial cavity is discussed and we confirm that the PCR assay using the previously described T. foetus-specific primers TFR3 and TFR4 could be a useful alternative method for the diagnosis of bovine trichomonosis.

Molecular phylogenetic position of the genera Stephanonympha and Caduceia (Parabasalia) inferred from nuclear small subunit rRNA gene sequences.

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.