Enterocytozoon bieneusi is a major zoonotic pathogen causing human microsporidiosis, with small ruminants serving as crucial reservoir hosts. Hu sheep and local goats are pillar livestock breeds in Zhejiang Province, yet no parallel comparative epidemiological data on E. bieneusi exist for sympatric small ruminants in southern Zhejiang, and the infection status in Hu sheep remains entirely uncharacterized. Here, a total of 297 fecal samples (159 Hu sheep, 138 goats) were collected from four administrative regions in southern Zhejiang from 2021 to 2023 and screened via nested PCR targeting the ribosomal internal transcribed spacer (ITS) region of the E. bieneusi genome. Overall, the total E. bieneusi prevalence reached 18.18%, and Hu sheep exhibited a significantly higher infection rate (24.53%, 39/159) than sympatric goats (10.87%, 15/138; χ² = 9.27, P = 0.002). Obvious geographical and age-dependent disparities were observed in Hu sheep, with pre-weaning lambs (< 3 months) showing the highest prevalence (60.42%), while adult sheep (> 1 year) had the lowest rate (4.48%). No significant age or regional differences were detected in goats. Five genotypes were identified from Hu sheep: CM7 (n = 25), BEB6 (n = 10), CHG1 (n = 2), Type IV (n = 1), and CHG5 (n = 1). Five genotypes were detected from goats: CHG1 (n = 9), BEB6 (n = 3), Type IV (n = 1), CHG3 (n = 1), and CYG-2 (n = 1). BEB6, Type IV, and CHG1 were shared by both hosts. All genotypes belonged to zoonotic Group 1 and Group 2. This study represents the first systematic parallel survey of E. bieneusi in cohabiting Hu sheep and goats in eastern China, identifies distinct host-specific infection patterns linked to divergent rearing systems, provides molecular evidence for potential cross-species transmission, and underscores the need for breed-tailored biosecurity strategies under a One Health framework.

Enterocytozoon bieneusi is a major zoonotic pathogen causing human microsporidiosis, with small ruminants serving as crucial reservoir hosts. Hu sheep and local goats are pillar livestock breeds in Zhejiang Province, yet no parallel comparative epidemiological data on E. bieneusi exist for sympatric small ruminants in southern Zhejiang, and the infection status in Hu sheep remains entirely uncharacterized. Here, a total of 297 fecal samples (159 Hu sheep, 138 goats) were collected from four administrative regions in southern Zhejiang from 2021 to 2023 and screened via nested PCR targeting the ribosomal internal transcribed spacer (ITS) region of the E. bieneusi genome. Overall, the total E. bieneusi prevalence reached 18.18%, and Hu sheep exhibited a significantly higher infection rate (24.53%, 39/159) than sympatric goats (10.87%, 15/138; χ² = 9.27, p = 0.002). Obvious geographical and age-dependent disparities were observed in Hu sheep, with pre-weaning lambs (1 year) had the lowest rate (4.48%). No significant age or regional differences were detected in goats. Five genotypes were identified from Hu sheep: CM7 (n = 25), BEB6 (n = 10), CHG1 (n = 2), Type IV (n = 1), and CHG5 (n = 1). Five genotypes were detected from goats: CHG1 (n = 9), BEB6 (n = 3), Type IV (n = 1), CHG3 (n = 1), and CYG-2 (n = 1). BEB6, Type IV, and CHG1 were shared by both hosts. All genotypes belonged to zoonotic Group 1 and Group 2. This study provides the first systematic parallel survey of E. bieneusi in sympatric Hu sheep and goats in eastern China. It identifies distinct host-specific infection patterns potentially associated with divergent rearing systems, provides molecular data consistent with potential inter-species circulation and shared environmental exposure, and highlights the need for breed-tailored biosecurity strategies within a One Health framework.