Genotyping of Enterocytozoon bieneusi in Denmark and Sweden: Is the outbreak-linked genotype C a rodent-adapted genotype? Evidence from rodents and wastewater

Wastewater can serve as a useful indicator of the presence of pathogens circulating in the human population, including zoonotic pathogens mainly adapted to rodents. Rodents are small mammals distributed worldwide and capable of inhabiting various ecosystems, including sewer systems. Enterocytozoon bieneusi is the main etiological agent of human microsporidiosis, a well-established zoonosis; however, little is known about the epidemiology of microsporidiosis in Scandinavia, except for two foodborne outbreaks reported in Sweden and Denmark, both of which were caused by genotype C. The aim of this study was to determine the presence and genetic diversity of E. bieneusi in wild rodent populations in Denmark and influent wastewater samples from wastewater treatment plants in Sweden. Samples were analysed using nested PCR targeting the ribosomal internal transcribed spacer (ITS) and flanking regions of E. bieneusi, followed by Sanger sequencing. Sampled species included Apodemus flavicollis, Apodemus sp., Apodemus sylvaticus, Micromys minutus, Microtus agrestis, Mus musculus, and Myodes glareolus, with an overall positivity rate of 25.0 % (95 % CI, 14.4-38.4) (14/56). Meanwhile, E. bieneusi was detected in 82.8 % (95 % CI, 64.2-94.2) (24/29) of the wastewater samples. Sequence analysis revealed the presence of two known (C, EBCMAP-032), and a novel genotype (DKE-1) in rodents, and eight established (C, CYG-4, D, EBCMAP-007, EBCMAP-031, EBCMAP-032, Type IV, WildBoar3) and four new genotypes (SweWW1-SweWW4) in Swedish wastewater. A relatively high frequency of genotype C was observed in this study, suggesting a zoonotic-and possibly rodent-adapted source of the previous foodborne outbreaks caused by this genotype in Sweden and Denmark.