A comparative evaluation of PCR- based methods for species- specific determination of African animal trypanosomes in Ugandan cattle

Background: In recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. Individual species-specific multi-copy trypanosome DNA sequences can be targeted to identify parasites. Highly conserved ribosomal RNA (rRNA) genes are also useful for comparisons between closely related species. The internal transcribed spacer regions (ITS) in particular are relatively small, show variability among related species and are flanked by highly conserved segments to which PCR primers can be designed. Individual variations in inter-species length makes the ITS region a useful marker for identification of multiple trypanosome species within a sample. Methods: Six hundred blood samples from cattle collected in Uganda on FTA cards were screened using individual species-specific primers for Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax and compared to a modified (using eluate extracted using chelex) ITS-PCR reaction. Results: The comparative analysis showed that the species-specific primer sets showed poor agreement with the ITS primer set. Using species-specific PCR for Trypanozoon, a prevalence of 10.5\% was observed as compared to 0.2\% using ITS PCR (Kappa = 0.03). For Trypanosoma congolense, the species-specific PCR reaction indicated a prevalence of 0\% compared to 2.2\% using ITS PCR (Kappa = 0). For T. vivax, species-specific PCR detected prevalence of 5.7\% compared to 2.8\% for ITS PCR (Kappa = 0.29). Conclusions: When selecting PCR based tools to apply to epidemiological surveys for generation of prevalence data for animal trypanosomiasis, it is recommended that species-specific primers are used, being the most sensitive diagnostic tool for screening samples to identify members of Trypanozoon (T. b. brucei s.l). While ITS primers are useful for studying the prevalence of trypanosomes causing nagana (in this study the species-specific primers did not detect the presence of T. congolense) there were discrepancies between both the species-specific primers and ITS for the detection of T. vivax.