Detection of Pentatrichomonas hominis DNA in biological specimens by PCR

AIM: To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens. METHODS: Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs. CONCLUSIONS: A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of P. hominis in vaginal specimens has not been reported before.     

The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus

Conserved sequence amplification (CSA) has been used to obtain sequence data for two glycosidase genes from the primitive eukaryote Tritrichomonas foetus. Few genes have been cloned from this organism, and there is little information concerning protein sequence. CSA is reliant on the use of database searches to identify short sequences of 3–9 amino acids conserved within a protein across a wide range of species. PCR primers are then constructed based on this sequence data and the DNA is amplified and sequenced. In the case of the β-galactosidase gene, N-terminal amino acid sequence data were used to construct a primer that replaced the upstream primer to ensure the amplified product was related to β-d galactosidase CSA was also applied to the gene encoding the enzyme β-N-acetyl-d-glucosaminidase from T. foetus, but in this case a segment of DNA was amplified, which, if correct, should contain a third conserved motif. The products of the CSA were sequenced, and the data obtained were compared to data in the SwissProt database. The results obtained suggest that this approach is useful for the cloning of genes to obtain novel sequence data from organisms where little genetic information is available.     

Lipid metabolism in mucous-dwelling amitochondriate protozoa

Entamoeba, Giardia, and trichomonads are the prominent members of a group known as 'mucosal parasites'. While Entamoeba and Giardia trophozoites colonise the small intestine, trichomonads inhabit the genitourinary tracts of humans and animals. These protozoa lack mitochondria, well-developed Golgi complexes, and other organelles typical of higher eukaryotes. Nonetheless, they have developed unique metabolic pathways that allow them to survive and multiply in the small intestine and reproductive tracts by scavenging nutrients from the host. Various investigators have shown that these protozoa are unable to synthesise the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources. Therefore, questions of how they transport and utilise exogenous lipids for metabolic purposes are extremely important. There is evidence suggesting that these parasites can take up the lipids and cholesterol they need from lipoprotein particles present in the host and/or in the growth medium. Studies also support the idea that individual lipid and fatty acid molecules can be transported without the help of lipoproteins. Exogenous phospholipids have been shown to undergo fatty acid remodelling (by deacylation/reacylation reactions), which allows these protozoa to alter lipids, bypassing the synthesis of entirely new phospholipid molecules. In addition, many of these amitochondriates are, however, capable of elongating/desaturating long-chain fatty acids, and assembling novel glycophospholipid molecules. In this review, progress in various aspects of lipid research on these organisms is discussed. Attempts are also made to identify steps of lipid metabolic pathways that can be used to develop chemotherapeutic agents against these and other mucosal parasites.     

Molecular phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.     

Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum: comparative proteolytic activity

At least four proteolytic activities were detected in the lysates of each of Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum. These were HPAase, a dithiothreitol-dependent activity on hide powder azure; AZCase, a dithiothreitol-dependent activity on azocasein; and two distinct activities towards peptide nitroanilide derivatives--one was optimally active at pH 7 and stimulated by dithiothreitol; the other had no dithiothreitol requirement and was highly active at pH 5. HPAase and AZCase were active over a broad pH range. Overall, with respect to these four activities, T. batrachorum and T. vaginalis were quite similar. In contrast, T. vaginalis and T. foetus differed from one another in several respects, notably the level of HPAase activity and the properties of the dithiothreitol-independent activity. Multiple bands of proteinase activity were demonstrated with each species after electrophoresis of parasite extracts on polyacrylamide gels containing denatured haemoglobin. They appeared optimally at acid pH and in the presence of dithiothreitol. The proteinase band patterns of T. foetus were similarly complex (at least six bands), whereas T. batrachorum gave a much simpler pattern (three bands). The sensitivities to proteinase inhibitors suggested that all the activities were due to cysteine proteinases. The results show that there are some similarities in the proteolytic activities of all three trichomonad species, and that the two parasites of the urinogenital tracts of mammals possess additional features in common.