Abstract
Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as ‘test-of-cure’ tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.
Highlights
Animal African trypanosomosis (AAT) is a neglected tropical disease caused by parasites of the Trypanosoma genus and mainly affects livestock in Sub-Saharan Africa
We describe the development of a Nb-based T. congolense specific heterologous sandwich ELISA and its translation into an lateral flow assays (LFAs)
We demonstrate that the Nb44/Nb42 pair recognises T. congolense pyruvate kinase (TcoPYK), thereby validating yet another glycolytic enzyme as a candidate biomarker
Summary
Animal African trypanosomosis (AAT) is a neglected tropical disease caused by parasites of the Trypanosoma genus and mainly affects livestock in Sub-Saharan Africa. The sensitivity and specificity of AAT diagnosis have greatly been improved by the development of DNA-based assays such as PCR and LAMP, which aim to amplify parasite DNA20–24. While such assays are highly reliable and have the potential of detecting an infection before the manifestation of pathology, their deployment in the field is generally difficult. We demonstrate that the Nb44/Nb42 pair recognises T. congolense pyruvate kinase (TcoPYK), thereby validating yet another glycolytic enzyme as a candidate biomarker Using these Nbs, we have developed antigen-based immunoassays in two formats: a heterologous sandwich ELISA and an LFA prototype. We present a preliminary evaluation of the performance of the LFA on plasma samples from experimentally infected cattle
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