New discovery and ultrastructural description of Dientamoeba fragilis cysts and the establishment of an animal model for their study

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Dientamoeba fragilis is a pathogenic protozoan parasite which causes diarrhoea and gastrointestinal disease in humans with a propensity for chronic infections. Although Dientamoeba was discovered over a century ago, its life cycle and mode of transmission are poorly defined. No cyst stage has been described in the scientific literature and no animal models were available for the further study of this parasite in the past. The clinical and pathologic features of Dientmaoebiasis, along with the pathogenic mechanisms of the disease and the nature of the host defence weren’t fully elucidated. In this study the in vitro culture for D. fragilis was established and further improved, which increased the trophozoite numbers to large and sufficient numbers for further study. A new overlay was designed for the in vitro culturing of D. fragilis trophozoites which is Earle’s balanced salt solution (EBSS) enriched with ferric ammonium citrate and cholesterol. The large trophozoite numbers obtained from the in vitro culture using this overlay enabled their use to inoculate experimental animals in order to develop an animal model. A rodent model was developed using BALB/C mice and rats to study the mode of transmission of this parasite, which remained a mystery in the past. This was an important step in this research as attempts to establish an animal model for this parasite have been unsuccessful in the past. Moreover, the animal models enabled us to fulfil three criteria of Koch’s postulates for D. fragilis. The most important finding of this study was the discovery of a cyst stage of D. fragilis, adding to the evidence on the mode of transmission of D. fragilis via cysts. Ultrastructural observations of the cysts were carried out in detail using transmission electron microscopy. These studies of cysts showed a clear cyst wall surrounding an encysted parasite. The cyst wall was double layered with an outer fibrillar layer and an inner layer enclosing the parasite. Hydrogenosomes, endoplasmic reticulum and nuclei were present in the cysts. Pelta-axostyle structures, costa and axonemes were identifiable and internal flagella were present. These cysts shared similar morphological characteristics to those of Giardia, Histomonas, which belong to the same family as D. fragilis showing its phylogenetic relationship with these parasites. This study provides additional novel details and knowledge of the ultrastructure of the cyst stage of D. fragilis, that plays an important role in the mode of transmission of this pathogen. The data support the pathogenic potential of this organism, demonstrates chronic infection and parasite carriage along with prolonged shedding of the organism. The recurrent nature of Dientamoebiasis in human hosts could be attributable to the cysts stage which is more resistant to the environmental conditions than the trophozoite stage. Further research is needed to study the biology and the virulence of the cyst stage of D. fragilis. The discovery of the cyst stage and the establishment of an animal model have major implications for the potential control of Dientamoebiasis in humans and in gaining a better understanding of the disease itself.
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