Improvement of diagnostics for Trypanosoma equiperdum infecting equines in South Africa

Abstract

Dourine is a sexually transmitted disease of equines caused by Trypanosoma equiperdum. Dourine has worldwide distribution and is an economically important veterinary disease. There is little to no active research on dourine in South Africa despite the high number of reported cases in various provinces. The OIE recommended diagnostic technique is a serological assay referred to as complement fixation test which confirms exposure to infection. The lack of simple and reliable diagnostic methods is an obstruction in the effective control of diseases. It is still difficult to entirely distinguish all Trypanozoon species. Therefore, the aim of this study was to develop DNA based diagnostic assays including conventional polymerase chain reaction (conPCR), real-time PCR (qPCR) and loop-mediated isothermal amplification (LAMP) for the detection of Trypanosoma equiperdum infections in South African equids. Primer sets and probes were designed from the repetitive insertion mobile element (RIME) gene. The three assays namely conPCR, qPCR and LAMP specifically amplified T. equiperdum DNA when tested against other parasites which co-infect equines. However, the specificity of qPCR was not stable and required analyses using melting curves. The detection limit of conPCR and LAMP for serially diluted DNA was 1×10-5 and 1×10-7 for conPCR and LAMP which is equivalent to 1 and 0.001 trypanosome cells/ml respectively, while the SYBR green and probe based qPCR had 1×10-5 detection limit which is equivalent to 1 trypanosome/ml. The conPCR, qPCR and LAMP assays were used to screen DNA extracted from blood collected from horses and donkeys in South Africa. The detection performance of LAMP was higher than that of real-time qPCR, conPCR with 70.8%, 52.1% and 62.5% respectively. Data obtained from this study show that LAMP, conPCR and qPCR assays can be a useful supplementary tools to clinical signs and microscopical diagnosis of T. equiperdum infections in equines in South Africa