Prevalence and genetic characterization of Cryptosporidium spp. and Enterocytozoon bieneusi in horses in central and western Inner Mongolia, China

Background Cryptosporidium spp. and Enterocytozoon bieneusi are important enteric pathogens infecting humans and a wide range of animal hosts, including horses, and some species and genotypes have zoonotic significance. However, information on their prevalence and genetic characteristics in horses in Inner Mongolia remains limited. This study investigated the prevalence, species/genotype composition, and genetic diversity of Cryptosporidium spp. and E. bieneusi in horses from central and western Inner Mongolia. Methods From May to September 2025, 325 horse fecal samples were collected from Otog Banner, Zhenglan Banner, and Abaga Banner, Inner Mongolia, China. Cryptosporidium spp. and E. bieneusi were detected by nested PCR targeting the SSU rRNA gene and ITS region, respectively. C. hominis -positive samples were further subtyped by gp60 amplification. Phylogenetic and haplotype analyses were performed to assess genetic relationships and diversity, and prevalence was compared among geographic, age, and health-status groups. Results The overall prevalence of Cryptosporidium spp. and E. bieneusi was 2.8% (9/325) and 1.5% (5/325), respectively. Two Cryptosporidium species were identified: C. hominis (55.6%, 5/9) and C. parvum (44.4%, 4/9). All C. hominis isolates belonged to the Ik subtype family, including IkA18G1 and IkA19G1. Cryptosporidium prevalence was significantly higher in foals than in adult horses (11.5% vs. 1.1%, p < 0.05) and in diarrheic than non-diarrheic horses (16.1% vs. 1.4%, p < 0.05). All E. bieneusi -sequences were identified as genotype horse1 and clustered within Group 1. Phylogenetic and haplotype analyses showed that the Cryptosporidium sequences were closely related to previously reported equid-derived sequences from China. C. hominis exhibited genetic diversity, whereas the E. bieneusi horse1 genotype showed a relatively conserved genetic structure. Conclusion This study provides new molecular epidemiological and genetic insights into Cryptosporidium spp. and E. bieneusi in horses in central and western Inner Mongolia, including, the first regional report of the C. hominis gp60 subtype IkA19G1. The higher Cryptosporidium prevalence in foals and diarrheic horses highlights these groups as priorities for surveillance. These findings provide baseline information for future molecular surveillance and assessment of potential public health risks associated with these enteric pathogens in horses in this important horse-breeding region of China.