Ultrastructural description of Dientamoeba fragilis and a new viral-like particle
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Dientamoeba fragilis is a trichomonad protozoan found in the gastrointestinal tract of humans and is implicated as a cause of diarrhoeal disease. Despite its widespread occurrence and associated symptoms, very little is known about the biology and pathogenicity of D. fragilis. Advances in cell biology of other trichomonads means there is a need to advance knowledge on this neglected protozoan. In this study, the morphological characteristics and ultrastructure of D. fragilis were described in detail using different microscopy techniques. Scanning electron microscopy, transmission electron microscopy, confocal and light microscopy were used to characterise D. fragilis populations growing in xenic culture. Under the SEM, two types of D. fragilis populations were identified based on cell surface structure: smooth cells and ruffled cells. Typically D. fragilis has a spherical or oval shape with a granular, vacuolated cytoplasm and some cells are amoeboid. Dientamoeba fragilis exhibited different motile forms with visible pseudopodia. The organelles in D. fragilis were analysed by transmission electron microscopy; the pelta, flagella, undulating membrane or pseudocyst-like forms were not found. The presence of hydrogenosomes in D. fragilis is described which has not been previously reported. The majority of cells grown in culture were mononucleate while most cells in permanent stained faecal smears were binucleate. Evidence is presented using confocal microscopy that the two nuclei of D. fragilis are identical in DNA content. In addition, the discovery of a virus-like particle is reported for the first time in D. fragilis. This study provides extensive and new detail on the ultrastructure of D. fragilis that is an emerging cause of human enteric disease. Dientamoeba fragilis virus (virus-like particles or VLPs) was studied further: it was approximately 33-40 nm in size and the most common shape was spherical. These VLPs contain an inner dark core surrounded by an electron-lucent layer and an electron dense capsid coat. Virus particles are found extensively in the perinuclear region of the trophozoite, and especially around microtubules and in association with the Golgi complex. Virus particles were also found in the vicinity of endoplasmic reticulum, axostyle, and near to the parabasal filament but no VLPs were found in the nucleus. Dientamoeba fragilis VLPs were also detectable in dying trophozoites present in in vitro cultures. Whether viral load contributes to cell death is not yet known. The identity of the D. fragilis viral genome was also studied. Several different extraction methods were screened and three different methods were optimized to identify dsRNA from Trichomonas vaginalis (B7268 isolate) which was used as a positive control for the isolation of viral dsRNA. These optimized methods were evaluated to identify D. fragilis viral genome. No viral RNA or dsRNA was isolated from D. fragilis suggesting that unlike T. vaginalis, D. fragilis trophozoites do not contain a dsRNA virus, or that the abundance of the virus was so low that it prevented the identification of viral nucleic acid. The epidemiology of D. fragilis has not been studied in detail and as a small side project I investigated hospital records for infections of children. Consequently, a case-controlled study was conducted to document the extent of D. fragilis infections in children presenting to a major Sydney Hospital. Treatment options are also discussed. In total, hospital data from 41 children were included in the study along with a control group. Results showed that diarrhoea (71%) was found to be the most common symptom followed by abdominal pain (29%). In addition, diarrhoea was statistically more significant in children with D. fragilis infection compared to a control group. In this study, the most common antimicrobial used for treatment was metronidazole (n=41), with complete resolution of symptoms and clearance of parasite occurring in 85% of cases. Moreover, a treatment failure rate of 15% was identified in children treated with metronidazole. These studies further suggest the pathogenic nature of D. fragilis and it is recommended that all laboratories must routinely test for D. fragilis as treatment which eliminates the parasite usually results in the resolution of symptoms. In summary, this thesis has discussed many novel aspects on the biology of D. fragilis and provide new knowledge on the cell biology of this protozoan and a new protozoan virus.
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