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Abstract: Pythium insidiosum, the aetiological agent of pythiosis, has been reported to cause disease in mammals. Although several aspects of this pathogen have been extensively investigated, its ultra-structural features and the location and characterization of the antigens expressed during infection have yet to be examined. During this study the ultrastructural characteristics and the mapping of P. insidiosum hyphal antigens from in vitro cultures were investigated. The ultrastructural study showed similarities between the hyphal features of this mammalian pathogen and other Pythium spp. Using immuno-electron microscopy and protein-A colloidal gold (PACG)-labelling, anti-P. insidiosum antibodies from the sera of infected hosts (bovine, canine, equine, feline, and human), were found to bind specifically to several cytoplasmic and cell wall antigens within the hyphae of P. insidiosum. The anti-P. insidiosum antibodies present in the sera from an infected feline showed only 85% gold binding, whereas the PACG particles failed to bind to the canine antibodies. The mapping of the hyphal antigens of P. insidiosum could be of importance for the specific selection of these antigens and their future molecular characterization. In addition, the antigens of P. insidiosum detected by sera from infected hosts could be used as purified antigens in the diagnosis and the immunotherapy of pythiosis. [Copyright &y& Elsevier]