Comparative genetic analysis of tritrichomonadid protozoa by the random amplified polymorphic DNA technique

Abstract

The taxonomic classification within the genus Tritrichomonas is a subject of controversy, and, potentially, separation of the tritrichomonads from cattle and swine on the species level is not valid. To tackle this hypothesis we comparatively assessed several isolates of protozoan parasites from the three Tritrichomonas species T. foetus, T. suis, and T. mobilensis by the RAPD (random amplified polymorphic DNA) technique. In this method with 20 different primers, all T. foetus and T. suis isolates resulted in identical genomic fingerprints, thus yielding additional evidence for the genetic identity of T. foetus and T. suis. In contrast, it turned out that the species T. mobilensis isolated from the squirrel monkey is genetically distinct and can clearly be discriminated from the other tritrichomonads. Consequently, the results obtained in this study support a possible future revision of the taxonomic classification of the genus Tritrichomonas.

Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites.

“…This branch again had a bootstrap value of 100 %. These results are in agreement with the results of other authors (Hammond & Leidl, 1957 ;Felleisen, 1998) (Kulda et al, 1986), but some results of karyological studies suggest the possible existence of meiosis in a fraction of Trichomonas vaginalis cells in in vitro cultures (Drmota & Kra! l, 1997).…”

Detection ofTritrichomonas Foetusby Polymerase Chain Reaction in Cultured Isolates, Cervicovaginal Mucus, and Formalin-Fixed Tissues from iNfected Heifers and Fetuses

“…Recently developed morphologic, metabolic, protein/antigenic, and genetic evidence suggests that T. foetus and T. suis are the same species. 11,12,14,25 Sequence similarity at or near 100% has been shown for the 2 protozoa at both the 16S ribosomal RNA gene and 5.8S-ITS regions, although other tritrichomonadid species, including Tritrichomonas mobilensis, also showed near identity at the 16S site, suggesting that this group of organisms may have recently evolved. 11,25 The positive PCR results for T. suis obtained in this study and others are consistent with identity between T. foetus and T. suis, although a larger number of isolates of each need to be tested before such an identity can be accepted.…”

“…16 Recently, a PCR assay that amplifies a DNA segment in the 5.8S ribosomal RNA and internal transcribed spacer (5.8S-ITS) region of the genome of laboratory strains of T. foetus was described. [11][12][13] This communication reports the use of such a T. foetus-specific PCR assay to test trichomonad isolates from laboratory and field sources and to test for the presence of T. foetus in cervicovaginal mucus (CVM) of experimentally infected heifers and in formalin-fixed tissues from T. foetus-infected fetuses and heifers.…”

“…An additional pair of less-specific primers (i.e., primers that amplify a portion of the 5.8S-ITS region in a wide variety of trichomonad species) can be used as a procedural control, i.e., it will be ''positive'' if there is DNA on the gel from essentially any type of trichomonad, whereas the 347-bp band will only be present when the true pathogen, T. foetus, is amplified with TFR3/TFR4. [11][12][13] This 2-step PCR was recently used to identify trichomonads isolated from the prepuces of bulls in Argentina as non-T. foetus trichomonads. 9 The PCR assay would seem to represent a significant improvement in diagnosis in the female, whose ability to clear the organism from the vagina within 6-12 weeks of infection can hamper herd-level diagnostic efforts, that is, trichomoniasis is often not suspected until the time of pregnancy diagnosis, typically 4-7 months after exposure, at which time culture of CVM is usually nonrewarding.…”

“…i Primer sequences used were primer TFR3, 5Ј-CGGGTCTTCCTATATGAGACAGAACC-3Ј, and primer TFR4, 5Ј-CCTGCCGTTGGATCAGTTTCGT-TAA-3Ј. [11][12][13] Reaction mixtures (50 l) contained the following: 5 l GeneAmp PCR 10ϫ buffer, 2 U Taq polymerase, 100 M of each deoxyribonucleotide, 10 pmol of each primer, and 3 l of the DNA sample. The PCR assay included 40 cycles of denaturing at 94 C for 30 seconds, annealing at 67 C for 30 seconds, extension at 72 C for 90 seconds, and final extension at 72 C for 15 minutes.…”