Molecular Detection of Enterocytozoon bieneusi in Free-Range Sheep and Domestic Dogs from the Greater Hinggan Mountains Area of China

<i>Enterocytozoon bieneusi</i> (<i>E. bieneusi</i>) is a globally distributed microsporidian that infects both humans and animals. However, reports on its occurrence in free-ranging sheep and domestic dogs from rural areas are limited. The objective of this study was to investigate the prevalence and genotype distribution of <i>E. bieneusi</i> among freely grazing sheep and household dogs in the Hinggan Mountain region of China. A total of 235 fecal samples were collected, consisting of 95 from sheep and 140 from dogs, and screened for <i>E. bieneusi</i> by nested PCR targeting the internal transcribed spacer region (ITS) within the nuclear ribosomal DNA (rDNA) gene. The total infection rate of <i>E. bieneusi</i> amounted to 3.0% (7/235), with sheep exhibiting a rate of 5.3% (5/95) and dogs demonstrating a rate of 1.4% (2/140). Sequence analysis identified two known genotypes in sheep (BEB6 and NESH4, both within phylogenetic group 2) and one known genotype in dogs (CHN-F1, phylogenetic group 1), with CHN-F1 being reported in dogs for the first time. Importantly, the detection of BEB6, a genotype previously associated with human infection, suggests that sheep could act as a potential reservoir contributing to zoonotic transmission and environmental contamination. These findings expand knowledge of the genotype spectrum of <i>E. bieneusi</i> in rural animal populations and provide evidence of possible cross-species transmission between humans and the surveyed animals in the region.