L E Eyssen1; J Viljoen1; R Peter2; P Büscher3; T Coetzer1;
1 Biochemistry, University of KwaZulu-Natal, South Africa; 2 Global Alliance for Livestock Veterinary Medicine, UK; 3 Institute of Tropical Medicine Antwerp, Belgium
DiscussionTsetse-transmitted haemoprotozoan parasites, Trypanosoma congolense and T. vivax, cause African animal trypanosomiasis (nagana), a wasting disease of cattle and small ruminants. Current tsetse control methods are not effective and drug resistance has been reported in 17 African countries. No new drugs have been developed for nagana in the past 60 years. The variable surface glycoprotein coat of the parasite makes vaccine development unlikely. A further impediment to nagana surveillance and control is the lack of a simple, rapid and cost-effective diagnostic test for use in resource poor endemic settings.
Current diagnostic tests are either based on detecting parasites in the blood of infected animals using microscopy, serodiagnostics using parasite lysates to detect antibodies or by amplifying parasite specific DNA; all of which lack specificity and are costly.
We identified cysteine and serine peptidases, which are released by trypanosomes in the host bloodstream, that have potential as diagnostic targets. Recognition of recombinantly expressed cathepsin L-like peptidases from T. congolense and T. vivax (TcCATL and TvCATL) and oligopeptidase BTcOPB and TvOPB) by sera of experimentally infected cattle was tested in an indirect ELISA. The best performing peptidase antigens were adapted to a penside/dipstick test format to detect antibodies in T. congolense and T. vivax infected cattle sera. Since antibody-detection tests cannot distinguish between current and past infections, an antigen detection test was devised based on OPB-specific single chain variable fragment (scFv) antibodies identified from the Nkuku® phagemid library. This OPB-specific scFv recognises a conserved peptide in the T. congolense and T. vivax OPB homologs, and detected native OPB in a western blot. An antigen detection ELISA using this scFv as capture antibody and rabbit-anti-OPB IgG-HRPO antibody showed that OPB levels fluctuated with parasitaemia in sera from T. congolense infected cattle.