Detection and molecular characterization of <i>Enterocytozoon bieneusi</i> , Microsporidia, in wild small mammals from Portugal

Enterocytozoon bieneusi is a widespread endoparasite, commonly found in the gastrointestinal tract of humans and various vertebrate and invertebrate animals. It is the most common cause of microsporidiosis in humans, particularly among immunosuppressed individuals. Given the limited epidemiological data in Portugal, this study aimed to estimate the occurrence of E. bieneusi, perform genetic characterization, and explore its circulation dynamics in small mammal populations of Trás-os-Montes region. A total of 294 faecal samples from rodents and shrews non-invasively collected were analysed using a nested polymerase chain reaction (PCR) assay targeting the internal transcribed spacer region and flanking ribosomal RNA coding sequences of E. bieneusi. The parasite DNA was detected in two rodent samples: wood mouse (Apodemus sylvaticus) and Lusitanian pine vole (Microtus lusitanicus), corresponding to an overall occurrence of 0.68%. Our findings represent the first report of E. bieneusi in Lusitanian pine vole and the first detection of this parasite in small mammals from Portugal. Both samples belonged to the zoonotic Group 1, with one identified as genotype C. These findings provide baseline data on the distribution and host range of E. bieneusi in Portuguese wildlife and may guide future surveillance efforts within a One Health framework.