BACKGROUND: Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are important zoonotic pathogens. In Inner Mongolia, a single pathogen molecular epidemiological survey of these three protozoa was previously conducted on only 176 fecal samples donkeys. In this study, the purpose is to simultaneously evaluate the zoonotic potential of three protozoa. METHODS: Small subunit ribosomal RNA (SSU rRNA) and gp60 from Cryptosporidium
SSU rRNA, gdh, tpi, and bg from G. duodenalis
and ITS of E. bieneusi were analyzed in 300 fecal samples collected from donkeys from two intensive farms in Inner Mongolia in 2022. Sample processing, nucleic acid extraction, polymerase chain reaction, sequencing, and sequence analysis were performed to determine the prevalence and genetic characteristics of the protozoans. RESULTS: Our results revealed that 75.33% (226/300) of the samples were infected with at least one protozoan, whereas 2.33% (7/300) were simultaneously infected with all three genera. Mixed infection was observed in 20.67% (62/300
Cryptosporidium + G. duodenalis), 5.33% (16/300
Cryptosporidium + E. bieneusis), and 5.67% (17/300
G. duodenalis + E. bieneusis) of the samples. Overall prevalence of Cryptosporidium, G. duodenalis, and E. bieneusi was 56.33% (169/300), 36.33% (109/300), and 13.33% (40/300), respectively. Five Cryptosporidium species were identified, with C. andersoni being the predominant species. The C. parvum subtypes identified were IIdA20G1 (66.67%) and IIdA19G1 (33.33%). Three G. duodenalis assemblages were identified, with assemblage B being predominant. Seven isolates formed two different assemblage B MLGs, two different assemblage A MLGs, and two different assemblage B + E MLGs. Three known and eight novel sequences of E. bieneusis were identified in group 1 (including horse1 and NMG6-NMG8) and group 2 (including J, BEB6, and NMG1-NMG5), with horse1 being the predominant genotype. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that donkeys can be infected with a combination of two or three protozoa, with C. andersoni being the most detected Cryptosporidium species. Meanwhile, assemblage B was predominant among G. duodenalis, whereas horse1 was the dominant genotype for E. bieneusi. Moreover, the prevalence of C. bovis, C. ryanae, C. suis, C. parvum subtype IIdA20G1 and eight novel sequences of E. bieneusis and new features of G. duodenalis assemblages were identified in donkeys for the first time. These findings reconfirmed that donkeys in Inner Mongolia are infected with these three protozoa, emphasizing the risk of disease transmission by these protozoans to humans.