Molecular epidemiology and cross-species transmission risk of Enterocytozoon bieneusi between humans and livestock: Evidence from Lishui, China

<i>Enterocytozoon bieneusi</i> is a zoonotic parasite with a broad host range and public health significance. In China, livestock production is predominantly small-scale, with cattle and sheep commonly maintained under extensive or semi-intensive husbandry systems that lack adequate biosecurity measures. Lishui, Zhejiang Province, typifies this model, where intensive and non-intensive farming systems coexist, and where abundant rainfall and dense water networks facilitate pathogen transmission. A total of 588 fecal samples were collected from cattle (<i>n</i> = 175), sheep (<i>n</i> = 228), and humans (<i>n</i> = 185) across nine counties in Lishui. Nested PCR targeting the <i>ITS</i> region was used for detection and genotyping, followed by phylogenetic and haplotype network analyses. The overall infection rates were 32.9 % in sheep, 4.5 % in cattle, and 1.6 % in humans, with all human cases occurring in occupationally exposed farm workers. Significantly higher infection rates were observed in intensively managed herds and in young animals under one year of age (<i>P</i> < 0.05). Five genotypes were identified in sheep, among which BEB6 was predominant (80.0 %), while cattle harbored genotypes BEB8 and J. Human isolates comprised genotypes BEB6, J, and I. Phylogenetic analyses placed all identified genotypes within Group 2, and haplotype network reconstruction revealed 10 haplotypes, some of which were shared between human and livestock samples from the same farms. These findings highlight cross-species transmission risks under current farming practices and underscore the necessity for One Health-based surveillance and control strategies.