Molecular identification of trichomonads in captive sugar gliders and tortoises with diarrhea: a case study

Trichomonads are widely distributed protozoa; however, studies on these organisms in captive sugar gliders and tortoises kept as companion animals remain limited. This study investigated trichomonads in four captive companion animals presenting with diarrhea-two sugar gliders (Petaurus breviceps), one red-footed tortoise (Chelonoidis carbonaria), and one Horsfield's tortoise (Testudo horsfieldii)-using conventional PCR and 18S rRNA gene metabarcoding. Light microscopic examination of fresh fecal samples at a veterinary clinic confirmed the presence of motile protozoa morphologically consistent with trichomonads in all animals. To identify the species, fecal samples were collected from each animal, and DNA was extracted for PCR and metabarcoding targeting the 18S rRNA gene. Trichomonads were not detected in any of the four samples by screening conventional PCR. In contrast, subsequent metabarcoding targeting the V9 region of the 18S rRNA gene successfully identified Trichomitus batrachorum in both tortoises and low abundance trichomonads in one sugar glider. Subsequent PCR using alternative primer sets confirmed the presence of trichomonads in all four animals. Phylogenetic analysis identified T. batrachorum in both tortoises, Simplicimonas sp. in one sugar glider, and Pentatrichomonas hominis in the other sugar glider. These findings expand current knowledge of the host range of trichomonads in captive sugar gliders and tortoises and suggest their possible clinical relevance in these animals presenting with diarrhea.