Prevalence and Molecular Characterization of Enterocytozoon bieneusi in Horses From Northern and Eastern China: Investigation of Associated Risk Factors and Assessment of Zoonotic Potential.

Enterocytozoon bieneusi is a zoonotic microsporidian threatening public health, yet epidemiological data on equine infections in China are scarce despite frequent human-horse contact. In this study, 412 fresh horse fecal samples were collected from four Chinese regions covering four rearing systems and were screened for E. bieneusi internal transcribed spacer (ITS) sequences via nested PCR, with risk factor and phylogenetic analyses conducted. The overall infection prevalence reached 15.0% (62/412), varying geographically from 9.7% (Wenzhou) to 29.3% (Qiqihar). Foals <1 year and male horses exhibited significantly higher infection risks, with crude prevalence ratios (PRs) of 4.47 and 2.28, respectively, while prevalence did not differ statistically across rearing modes (p = 0.182). Eighteen genotypes were identified: 11 known (D, horse1, Peru8, Type IV, EbpC, CAF1, GX3, HNR-VI, SHWR5, SYSWR18, SYSWR23) and seven novel (HRBM1, KDM1, KDM2, QQHRM1, QQHRM2, QQHRM3, WZM1). Genotype D was dominant, accounting for 58.1% (36/62) of positive isolates, followed by genotype horse1 at 14.5% (9/62) and genotype EbpC at 3.2% (2/62). The other eight known genotypes and all seven novel genotypes each had one isolate. All seven novel genotypes clustered within zoonotic Group 1 with all the known genotypes except HNR-VI identified here. This study confirms widespread equine E. bieneusi circulation across eastern and northern China, modulated by geography, age, and sex. The predominance of zoonotic genotypes implies horses serve as potential reservoirs for human transmission, supplying baseline epidemiological evidence for equine One Health biosecurity management.