Molecular and parasitological survey of trypanosomosis in KwaZulu-Natal and the development of cathepsin L-like real-time PCR assays for parasite discrimination

Abstract

African animal trypanosomosis (AAT), is an infectious parasitic disease of wildlife and livestock caused by multiple species and strains of Trypanosoma. In South Africa, it is restricted to northern KwaZulu-Natal (NKZN) and caused by Trypanosoma congolense and Trypanosoma vivax. A cross sectional study was conducted to determine AAT prevalence in 384 goat samples and identify trypanosome species circulating in 60 cattle at diptanks that are on the interface with Hluhluwe-uMfolozi game reserve in NKZN. Both cattle and goat samples were analysed using the buffy coat technique (BCT) and a polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 1 (ITS) region. Cattle samples were further analysed using an ITS quantitative real-time PCR (qPCR) assay designed for the detection of T. congolense, T. vivax and Trypanosoma brucei. None of the goat samples tested positive for Trypanosoma infections. The ITS qPCR assay detected Trypanosoma DNA in 30% of the cattle samples, while only 8.3% were positive with the ITS PCR and 11.7% were positive using BCT. Quantitative real-time PCR assays were designed to amplify a 98 base pairs (bp), 137 bp and 116 bp fragment of the cathepsin L-like (CATL) gene from T. brucei, Trypanosoma theileri and T. congolense, respectively. Each assay was shown to be efficient (>94%) and specific (109 to 102/101 copies/reaction) in the detection of Trypanosoma species. The diagnostic efficiency of the CATL qPCR assays was further tested using 60 cattle and 39 goats’ blood samples collected at three diptanks in NKZN. The CATL qPCR assays detected T. congolense and T. theileri infections in 33.3% of the cattle samples. The CATL qPCR assays also detected T. congolense infections in goats (23.1%) that were neither detected by BCT nor the ITS PCR. The CATL qPCR assays provide an additional, sensitive and specific tool for Trypanosoma diagnostics. The presence of trypanosomes in goats suggests they might be potential reservoirs of infections to other livestock.