Molecular Detection and Characterisation of Entamoeba species in Australian Cane Toads (Rhinella marina) and Australian Native Snakes

Abstract

One of Australia’s infamous pest, cane toads, were found to be infected with potentially pathogenic protozoan parasite within the genus of Entamoeba. It was reported that a number of cane toads in Northern Territory population was presented with gastrointestinal disease and therefore is the first agent of disease to be described in cane toads, causing morbidity and mortality. With its invasion dynamic, ecological relationship, and impact of cane toads on Australian native fauna still under evaluation, first report of entamoebiasis in cane toads is an emerging wildlife disease. This thesis is a part of investigation into the significance and impact of novel Entamoeba sp. CT1 and Entamoeba ranarum found in Australian native fauna and cane toad populations. This thesis focuses on two aspects: one is on the technical aspect of diagnostic of Entamoeba species of cane toads and native fauna, and the other is on the molecular analysis of the SSU-rRNA gene for identification and taxonomy of Entamoeba. This thesis evaluates the application of various methods of diagnostic of Entamoeba in faecal and colon of wild cane toads. Methods evaluated include direct wet mount, histology, conventional PCR and real-time PCR, where recommendations on future clinical diagnosis and epidemiological studies were made based on this evaluation. In order to investigate the distribution of Entamoeba in wild native fauna, non-invasive faecal detection of Entamoeba was implemented onto native snake populations that share prey and predatorial relationship with cane toads. Steps taken to obtain and analyse complete sequences (SSU-rRNA gene) of Entamoeba species was described and used to infer evolutionary relationship of the Entamoeba species of interest. Comparative data on performance of each diagnostic method for detection of Entamoeba (colon and faecal) in cane toads were obtained. For the purpose of non-invasive method of diagnosis (faecal detection), this thesis provided evidence for the use of molecular-based assays (real-time PCR and conventional PCR) as the recommended approach. By implementing this approach, this study was able to provide firsthand evidence for the presence of Entamoeba ranarum in faecal samples of wild keelback snakes. Molecular analyses of 18S rRNA gene of Entamoeba species in cane toads and snakes placed the two species well into the amphibian/reptilian clade and further expand the knowledge on Entamoeba genetic diversity