Abstract
Enterocytozoon bieneusi (E. bieneusi) 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 E. bieneusi 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 E. bieneusi by nested PCR targeting the internal transcribed spacer region (ITS) within the nuclear ribosomal DNA (rDNA) gene. The total infection rate of E. bieneusi 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 E. bieneusi in rural animal populations and provide evidence of possible cross-species transmission between humans and the surveyed animals in the region.