Enterocytozoon bieneusi is an obligate intracellular protozoan parasite that infects a wide range of mammal hosts and birds. Previous genotypic surveys were limited to measure the polymorphisms at the ribosomal internal transcribed spacer (ITS) that evolved slowly. Data on population structure are available only on E. bieneusi isolates from primates. This study explored the genotypic and phylogenetic characteristics of four mini- and microsatellites and performed a population genetic analysis in 39 E. bieneusi isolates of potentially zoonotic ITS genotype D from farmed foxes and raccoon dogs in China. Sequence polymorphisms facilitated determination of six, two, four, and five genotypes at markers MS1, MS3, MS4, and MS7, respectively. Patterns of phylogeny revealed different levels of diversity within and among the genetic markers. Clear genotypic and phylogenetic divergences between E. bieneusi isolates of ITS genotype D from fur animals and humans were observed at individual markers. Complete linkage disequilibrium and very limited recombination in subsequent population genetic analysis supported a clonal structure for E. bieneusi population from fur animals (FID). Phylogenetic analysis, genetic network, and measures of FST and gene flow demonstrated population differentiation of FID from two known human E. bieneusi populations HID (with a clonal structure) and HIA (with an epidemic structure). The data indicated an ideal resolving power of MLST compared to the previously widely used ITS genotyping and confirmed the clonal nature and population differentiation of E. bieneusi in various hosts.
