First Report of Molecular Prevalence and Genetic Characterization of Enterocytozoon bieneusi in Young Dairy Cattle in Western Romania

Enterocytozoon bieneusi is an important microsporidian pathogen with a broad host range and potential zoonotic relevance, yet molecular data from cattle in Romania are lacking. This study investigated, for the first time, the molecular prevalence, genotype diversity, and phylogenetic relationships of E. bieneusi in young dairy cattle in western Romania. A total of 190 fecal samples were collected from young dairy cattle, comprising pre-weaned calves and post-weaned cattle up to 12 months of age from 28 farms in Arad and Timiș counties and examined by nested PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. E. bieneusi DNA was detected in 27/190 cattle, corresponding to an overall prevalence of 14.2% (95% CI: 10.0–19.9%). Pre-weaned cattle showed a higher prevalence than post-weaned animals (18.1% vs. 8.1%), but the difference was not statistically significant (OR = 2.51, 95% CI: 0.96–6.54; p = 0.058). High-quality ITS sequences were obtained from 20 positive samples, revealing five established genotypes, namely BEB4 (45.0%), I (25.0%), J (10.0%), BEB8 (10.0%), and BEB17 (10.0%), among the genotyped isolates (n = 20). Phylogenetic analysis placed all Romanian isolates within Group 2, predominantly associated with ruminants but also containing genotypes reported in humans. These findings establish the first molecular baseline for E. bieneusi in young dairy cattle in western Romania and provide initial information on their epidemiological relevance within a One Health framework.