Elsevier

Experimental Parasitology

Volume 99, Issue 4, December 2001, Pages 181-189
Experimental Parasitology

Regular Article
Trypanosoma evansi: Cloning and Expression in Spodoptera fugiperda Insect Cells of the Diagnostic Antigen RoTat1.2

https://doi.org/10.1006/expr.2001.4670Get rights and content

Abstract

Urakawa, T., Verloo, D., Moens, L., Büscher, P., and Majiwa, P. A. O. 2001. Trypanosoma evansi: Cloning and expression in Spodoptera fugiperda insect cells of the diagnostic antigen RoTat1.2. Experimental Parasitology99, 181–189. A complementary DNA encoding the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigenic type (RoTat)1.2, currently used for experimental serological diagnosis of T. evansi infection in livestock, was cloned as a recombinant plasmid and sequenced. A recombinant baculovirus containing the coding region of RoTat1.2 VSG was constructed to express the protein in Spodoptera fugiperda insect cells. From this, sufficient quantities of the recombinant protein are being produced for empirical and wide-scale objective assessment of the diagnostic potential of this antigen. The gene encoding the RoTat1.2 VSG was shown by PCR to be present in the genomes of many different cloned isolates of T. evansi, but not T. brucei, from geographically separate regions of Africa, Asia, and South America. With the recombinant RoTat1.2 at hand, it is now possible to investigate the extent to which epitopes on this VSG are conserved among different T. evansi isolates.

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      Given the low sensitivity of parasitological examinations in chronic infections, DNA amplification techniques, such as the polymerase chain reaction (PCR), are often applied as surrogate (Büscher, 2014). For surra, Trypanozoon-specific primers targeting satellite DNA or ribosomal DNA are the most sensitive (Gari et al., 2010; Masiga et al., 1992; Njiru et al., 2005) while the distinction between T. evansi type A and type B can be made with PCRs specific for the type A RoTat 1.2 gene and specific for type B minicircles (Claes et al., 2004; Njiru et al., 2006; Urakawa et al., 2001). In addition to parasitological or molecular diagnostics, serological tests are useful to provide indirect evidence of the presence of T. evansi in a susceptible population or individual.

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      Several molecular diagnostics (Sengupta et al., 2010; Rudramurthy et al., 2013) and serological tests have been developed for the detection of trypanosomosis in animals which include, Suratex (Nantulya, 1994), CATT/T. evansi (Bajyana Songa and Hamers, 1988), LATEX/T. evansi, ELISA/ T. evansi (Verloo et al., 2000). Moreover, an attempt has been made by several research groups to improve serological (antibody detection) diagnostics using recombinant surface proteins such as variable surface glycoprotein (VSG) (Sengupta et al., 2012, 2014,2016; Urakawa et al., 2001), invariant surface glycoprotein (ISG) (Tran et al., 2008; Rudramurthy et al., 2015, 2017a,b) and flagellar protein (Ligi et al., 2016). The antibody detecting serological technique may not always detect true positive cases, because antibodies which are not found early in the infection may persit even after recovery or chemotherapy.

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    1

    Present address: London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.

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