Analysis of Trypanosoma equiperdum Recombinant Proteins for the Serological Diagnosis of Dourine

Abstract

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.

Keywords: ELISA; dourine; immunoblotting; recombinant proteins.

Figure 1

SDS-PAGE and Coomassie stain of purified proteins A0A1G4I8N3, A0A1G4I464 and A0A1G4I740 (GenScript) Lane M: molecular weight standard (Novex Sharp Prestained Protein Standard, Life Technologies); lane 1: protein A0A1G4I8N3 (the band of 9 kDA is a fragment of the protein A0A1G4I8N3 containing the His-tag as declared by the analysis report provided by GenScript); lane 2: protein A0A1G4I464; lane 3: protein A0A1G4I740.

Figure 2

(a) Western blotting test using T. equiperdum OVI whole antigen (lane 1) and recombinant proteins A0A1G4I8N3 (lane 2), A0A1G4I464 (lane 3) and A0A1G4I740 (lane 4) incubated with the reference horse serum positive for T. equiperdum (batch 019/1994, IZSAM); (b) T. equiperdum OVI whole antigen (lane 1) and recombinant proteins A0A1G4I8N3 (lane 2), A0A1G4I464 (lane 3) and A0A1G4I740 (lane 4) incubated with the reference horse serum negative for T. equiperdum. Lane M: molecular weight standard (Novex Sharp Prestained Protein Standard, Life Technologies).