Development of Cathepsin L-like Real-Time PCR Assays for the Detection of African Animal Trypanosomosis (AAT) in South Africa.

Samantha Mnkandla,Ilse Vorster,Luis Neves,Raksha Vasantrai Bhoora

doi:10.3390/pathogens11020136

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 done to determine AAT prevalence in 384 goat samples and identify trypanosome species circulating in 60 cattle at dip tanks that are on the interface with the Hluhluwe-uMfolozi game reserve in NKZN. Both cattle and goat samples were analyzed 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 analyzed using an ITS quantitative real-time PCR (qPCR) assays designed for the detection of T. congolense, T. vivax, and T. 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 bp, 137 bp, and 116 bp fragment of the cathepsin L-like (CATL) gene from T. brucei, T. 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 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.

Highlights

African animal trypanosomosis (AAT) is a debilitating disease of livestock caused by extracellular flagellate protozoan parasites belonging to the family Trypanosomatidae [1]
Using the buffy coat technique (BCT), 7 (11.7%) of the 60 cattle were positive for trypanosomes and only T. congolense was detected
The highest number of infections were recorded at Mvutshini and Ekuphindisweni, where 3 (15%) Trypanosoma-positive animals were detected at both dip tanks

Summary

Introduction

African animal trypanosomosis (AAT) is a debilitating disease of livestock caused by extracellular flagellate protozoan parasites belonging to the family Trypanosomatidae [1]. Three species of Trypanosoma endemic to Africa are Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei [1]. The detection of trypanosomes is determined by either observing the clinical signs of animals, conducting microscopic analysis, performing serological diagnosis, or using molecular tests such as the Loop-mediated isothermal amplification (LAMP) assay, Low-flow assay, and polymerase chain reaction (PCR) assays [9,10]. Molecular diagnostic techniques such as PCR assays have better sensitivity and specificity compared to parasitological techniques [11,12,13]. Due to their high sensitivity, quantitative realtime PCR assays allow for better detection of trypanosomes compared to parasitological techniques and conventional PCR assays [14,15,16]

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