Molecular epidemiology of Enterocytozoon bieneusi in humans and domestic animals from Heilongjiang province, China: high animal prevalence, novel genotypes, and possible zoonotic implications in rural households.

INTRODUCTION: Enterocytozoon bieneusi is a zoonotic parasite causing diarrheal disease in rural settings with frequent human-animal contact. METHODS: We conducted a cross-sectional study in Heilongjiang, China, enrolling 69 households across 10 villages. Humans and domestic animals were sampled. Nested PCR targeting the ITS region was performed. Genotypes were determined by sequencing, and genetic diversity was assessed using DnaSP. RESULTS: Human prevalence was 11.0% (13/118), while overall animal prevalence was 23.4% (162/693), with sheep being the highest (66.7%), followed by pigs (46.1%) and geese (20.5%). Prevalence was significantly higher in mammals than poultry (32.9% vs. 13.9%; p < 0.001). Sequence analysis yielded 29 distinct genotypes (10 known, 19 novel), with EbpC (36.0%), BEB6 (13.7%), EbpA (13.7%), and D (11.4%) being the most prevalent, most clustering within zoonotic Group 1. DISCUSSION: Notably, zoonotic genotypes EbpC and EbpA were identified in chickens and geese, extending the known host range of these genotypes. Human-animal genotype sharing was observed in only one household (genotype EbpC between humans, pigs, chickens, and geese), while intraspecies circulation and occasional animal-animal genotype sharing were more common. Phylogenetic and network analyses suggested localized diversification, though single-locus ITS limits definitive evolutionary inference. E. bieneusi is highly prevalent in rural domestic animals in Heilongjiang, with genetically diverse and potentially zoonotic genotypes circulating in livestock and poultry. Although household-level human-animal transmission appears limited in this dataset, the presence of shared zoonotic genotypes highlights the need for expanded surveillance incorporating environmental sampling and multi-locus typing to clarify transmission pathways and inform One Health strategies.