Molecular surveillance of Vittaforma-like microsporidia by a small-volume procedure in drinking water source in Taiwan: evidence for diverse and emergent pathogens

Viaaforma corneae belongs to microsporidia, which include over 1500 species of opportunistic obligate intracellular fungi infecting almost all known animal taxa. Although outbreaks of ocular infections caused by waterborne V. corneae have been reported in recent years, little is known about the occurrence of this pathogen in aquatic environments. In this study, 50 water samples from rivers and reservoirs around Taiwan in two seasons were analyzed to explore the presence of this pathogen in natural aquatic environments. A high detection rate of Waafinna-like amplicons (94%; 47/50) was observed in the water samples when examined by nested PCR with primer pairs specific to the small ribosomal subunit (SSU) rRNA gene. After electrophoresis. many lanes showed multiband patterns with expected molecular weights. After confirmation by DNA sequencing and by sequence alignment in the NCBI database, we identified a variety of Witaforma-like microsporidia Aith weak sequence similarity, with approximately 85% identity to V. corneae, thus indicating high diversity of microsporidia in aquatic environments. Phylogenetic analysis showed clear-cut microsporidian Glade classification and indicated that the most Viliafinna-like microsporidia in this study belong to Glade IV and cluster into four major groups. The first group is similar to the microsporidia associated with ocular microsporidiosis. The second group is associated with the diarrheal pathogens, whereas the third and fourth groups are a novel group and a zoonotic group, respectively. This study provides abundant sequencing infonnation, which will be useful for future molecular biological studies on microsporidia. Because microsporidia are important pathogens of animals and humans, it is urgently necessary to determine via a survey whether there are species with potential threats that have not yet been revealed.