Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections

Abstract

BACKGROUND: Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). RESULTS: This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. CONCLUSION: PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.

Figure 1

PCR specificity results for the different Trypanosoma (T.) species and subspecies in this study. Lane 1 pos. control RoTat 1.2, Lane 2 neg. control, Lanes 3–8 (T. evansi) are, respectively, AnTat 3.1, STIB 816, Zagora I.17, Colombia, Merzouga 56, CAN 86 K; Lanes 9–17 (T. equiperdum) are, respectively, AnTat 4.1, BoTat 1.1, OVI, STIB 818, Alfort, Hamburg, SVP, Am. Strain, Can. Strain ; Lanes 18–23 (T.b.brucei) are, AnTat 1.8, AnTat 2.2, AnTat 5.5, KETRI 2494, TSW 196, STIB 348; Lanes 24–31 (T.b.gambiense) are, respectively, AnTat 9.1, AnTat 11.6, AnTat 22.1, NABE, SEKA, ABBA, LIGO, LiTat 1.6; Lanes 32–36 (T.b. rhodesiense) are STIB 884, STIB 850, AnTat 25.1/S, Etat 1.2/S, AnTat 12.1/S ; Lanes 37–38 (T. congolense) are IL1180, TRT 17; Lane 39 (T. vivax) is ILRAD 700 and Lane 40 (T. theileri) is MELSELE ; Lanes M 100 bp molecular marker (MBI Fermentas, Germany).

Figure 2

Analytical sensitivity of the RoTat 1.2 PCR. Lanes M 100 bp molecular marker (MBI Fermentas, Germany); lane 1: 10⁵ trypanosomes, lane 2: 10⁴ trypanosomes, lane 3: 10³ trpyanosomes, lane 4: 10² trypanosomes, lane 5: 10 trypanosomes, lane 6: 1 trypanosome, lane 7: 0.1 trypanosome, lane 8: negative control.