Molecular characterization and high prevalence of Tritrichomonas foetus in bulls from the North-West Province of South Africa using real - time polymerase chain reaction (PCR) and conventional PCR diagnostics

Background and Aim: Bovine trichomonosis, caused by Tritrichomonas foetus, is a significant reproductive disease that impacts cattle productivity and breeding efficiency. In South Africa, routine diagnostic methods often depend on culture and microscopy, which may not accurately distinguish T. foetus from nonpathogenic trichomonads. This study aimed to determine the prevalence of T. foetus in bulls from the Dr. Segomotsi Ruth Mompati (DSRM) District, North-West Province, South Africa, using advanced molecular diagnostics, including real-time polymerase chain reaction (RT-PCR), conventional PCR, DNA sequencing, and phylogenetic analysis. Materials and Methods: A total of 239 sheath wash samples were collected between June 2018 and October 2020. Of these, 51 culture-positive trichomonad isolates were selected for molecular analysis. Microscopy and modified Giemsa staining were used to characterize protozoal morphology. DNA was extracted and subjected to RT-PCR with 5’ TaqMan™ probes, as well as conventional PCR targeting the 5.8S rRNA/Internal Transcribed Spacer (ITS) regions. PCR amplicons were sequenced, and phylogenetic trees were constructed using MEGA (maximum-likelihood, 1,000 bootstrap replicates). Statistical comparisons between diagnostic methods were performed using Chi-square and Cochran’s Q test. Results: RT-PCR detected T. foetus in 80.4% (41/51) of the culture-positive samples, with most isolates showing low Ct values, indicating strong positivity. Conventional PCR successfully amplified 12 isolates (300–340 bp), all of which were confirmed as T. foetus by sequencing. Phylogenetic analysis showed that the isolates clustered with the Southern African genotype, exhibiting 77%–87% similarity to Namibian strains and were closely related to Australian and Turkish isolates. No significant correlation was found between geographic location and PCR positivity. RT-PCR demonstrated significantly higher sensitivity than conventional PCR (p < 0.05). Conclusion: This study confirms a high prevalence of T. foetus in bulls in the DSRM district and demonstrates the superior accuracy of molecular diagnostics compared with culture and microscopy. The identification of genotypes closely related to Southern African strains highlights potential transboundary spread. Incorporating PCR-based screening into routine surveillance is essential for accurate diagnosis, minimizing unnecessary culling, and enhancing reproductive herd health. Further longitudinal studies are recommended to assess disease dynamics and inform regional control programs. Keywords: Tritrichomonas foetus, RT-PCR, conventional PCR, phylogeny, bovine trichomonosis, South Africa.