The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum

Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T. equiperdum. An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum. The luciferase assay would be suitable for the mass screening of chemical libraries against T. equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains. Keywords: Colorimetric assay, Drug screening system, Liquid culture, Luciferase assay, Trypanosoma equiperdu

Additional file 3: Figure S1. of Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia

The phylogenetic tree of the 18S rRNA and ITS region. A phylogenetic analysis was performed using the T. equiperdum IVM-t1, SITB818, STIB841, STIB842, BoTat1.1, T. evansi Tansui (Accession No. D89527.1), Cairo (AB551922.1), KAI.2 (AY912277), Sam.2 (AY912279.1), T. brucei TREU927 (AC012647), T. b. gambiense DAL972 (FN554966.1), T. b. gambiense Tsuua (AJ009141) and T. b. rhodesiense Utro (AJ009142) sequences. A: A phylogenetic tree based on the 18S rRNA sequence. B: A phylogenetic tree based on the ITS sequence. Figure S2. The maxicircle PCR of the Trypanozoon species. Gel electrophoresis images of the PCR products are shown in A to G, NADH-dehydrogenase subunit 7 (NAD7; 383 bp), Cytochrome oxidase subunit 2 (Cox2; 1747 bp), ATOas subunit 6 (A6; 299 bp), 12S ribosomal RNA (12S rRNA; 1597 bp in T. b. brucei GUTat3.1 strain and T. equiperdum STIB818 strain, 1415 bp in T. equiperdum STIB841, STIB842, BoTat1.1 strains, respectively), NADH-dehydrogenase subunit 7-cytochromeB (ND7-CyB; 1450 bp), Maxicircle unknown reading frame-NADH dehydrogenase subunit 1 (MURF-ND1; 1779 bp) and Maxicircle unknown reading frame 2-cytochrome oxidase subunit 1 (MURF2-Cox1; 1551 bp), respectively. M: the 100 bp and 1 kbp DNA ladders; Lanes 1 to 8 show T. b. brucei GUTat3.1, T. evansi IL3960, T. equiperdum IVM-t1, STIB818, STIB841, STIB842, BoTat1.1 strains and negative control (distilled water), respectively. (PPTX 164 kb