Detection and identification of Tetratrichomonas in a preputial wash from a bull by PCR and SSCP

Motile Tritrichomonas foetus-like trichomonads were found during microscopic examination of a wet mount sample of a preputial wash collected from a bull. Staining of the organisms with a modified Wright-Giemsa stain revealed that several had four anterior flagella of unequal length instead of the three anterior flagella of equal length characteristic of T. foetus. Limited propagation of these organisms was achieved in InPouch® medium but no growth occurred in modified Diamond's medium. The 5.8S rRNA gene and the flanking internal transcribed spacers were amplified from the trichomonad gDNA of two preputial wash samples and a fecal sample taken from the affected bull. Amplicons were subjected to single-strand conformation polymorphism (SSCP) analyses. The SSCP banding patterns of the amplicons from gDNA of the preputial wash samples were different from those of a T. foetus control sample. These unknown trichomonads were not detected in the fecal sample. The gDNA extracted from preputial washes was also subjected to PCR using primers developed to amplify the 16S rDNA of the non-T. foetus trichomonads, Tetratrichomonas and Pentatrichomonas spp. Amplicons were produced from the gDNA of the two preputial washes but not from the T. foetus gDNA control sample. The 16S rDNA sequences obtained from the trichomonads in the two preputial washes samples were 100% similar to that of a Tetratrichomonas species previously isolated from an Angus bull from the United States.