Prototheca bovis, a unicellular achlorophyllous trebouxiophyte green alga in the healthy human intestine.

Introduction. Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely. Hypothesis/Gap statement. There is an absence of detailed characterization of human Prototheca isolates. Aim. The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool. Methodology. Prototheca was isolated from faecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b ( cytb ) was also performed. Results. Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis . More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead. Conclusion. The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca . These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations.