First molecular detection and genotyping of Enterocytozoon bieneusi (Microsporidia) in fattening pigs from Italy

Background: Enterocytozoon bieneusi is the most reported microsporidian species in humans worldwide, with high prevalences also documented in animals, particularly pigs. Due to limited information on E. bieneusi in Italian livestock, this cross-sectional observational study aimed to investigate its prevalence, genetic diversity, and associated risk factors in pigs. For this purpose, 440 faecal samples were collected from pigs raised on 22 intensive farms in northern Italy during two sampling sessions, corresponding to the beginning and the end of the fattening cycle. A nested PCR protocol was performed to amplify the internal transcribed spacer region (ITS), followed by DNA sequencing for genotype identification. Moreover, several risk factors were evaluated as predictors of E. bieneusi positivity using generalised linear models. Results: In the first session, the positive samples were 197 out of 220 (89.5%); in the second, the positive samples were 82 out of 220 (37.3%). Overall, 279 out of 440 pig faecal samples (63.4%) were positive for E. bieneusi. In the multivariate analysis, age and faecal soiling of the animal’s body were significant risk factors (p-value < 0.05), with younger animals and those with a high degree of faecal soiling showing higher prevalences. All identified ITS sequences clustered within Group 1, which comprises the main genotypes with zoonotic potential. Conclusions: This study demonstrates a high prevalence of E. bieneusi in Italian pigs, albeit in a single production category, supporting their role as natural reservoirs. Further research is needed to assess the parasite’s presence in other pig categories, humans, environmental matrices, and food products to clarify its public health significance.