Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia

Abstract

Background: Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection. Methods: Trypanosomes isolated from the urethral tract of a stallion with suspected dourine, were directly cultivated using soft agarose media at 37 °C in 5 % CO2. For molecular characterization, 18S ribosomal RNA (rRNA) gene, the internal transcribed spacer (ITS) and 8 maxicircle DNA regions were amplified by a PCR and their sequences were determined. To analyze the ratio of the kinetoplastic/akinetoplastic population, the kinetoplasts and the nuclei of trypanosomes were subjected to Hoechst staining and observed by fluorescence microscopy. Results: In addition to the clinical symptoms and the molecular diagnosis, this stallion was definitively diagnosed with dourine by the detection of trypanosomes in the urethral mucosa. These results strongly suggested that the isolated trypanosome was true T. equiperdum. T. equiperdum isolated from the urethral tract was adapted in vitro using soft agarose media. Based on the results of a phylogenetic analysis of 18S rRNA and ITS, this T. equiperdum isolate was classified into the Trypanozoon clade. In a PCR of the maxicircle DNA region, only NADH-dehydrogenase subunits 4 and 5 was amplified. Clear kinetoplasts were observed in most of the T. equiperdum isolates. In contrast, most cultureadapted T. equiperdum were of the akinetoplastic form. Conclusion: We concluded that our isolated trypanosome was the first confirmed case of T. equiperdum in Mongolia and named it “T. equiperdum IVM-t1”. T. equiperdum IVM-t1 was well adapted and propagated in soft agarose media, which indicates that this culture method is useful for isolation of T. equiperdum from horses with dourine. Keywords: Dourine, In vitro culture, Maxicircle DNA, Mongolia, Soft agarose media, Trypanosoma equiperdum Abbreviations: 18S rRNA, 18S ribosomal RNA; ALP, Alkaline phosphatase; ALT, Alanine transaminase; AST, Aspartate aminotransferase; BUN, Blood urea nitrogen; CFT, Complement fixation test; CSF, Cerebrospinal fluid; ELISA, Enzymelinked immunosorbent assay; HCT, Hematocrit; HGB, Hemoglobin; HMI-9, Hirumi’s modified Isocove’s medium-9; ICT, Immunochromatographic test; ITS, Internal transcribed spacer; kDNA, Kinetoplast DNA; MCH, Mean corpuscular hematocrit; MCHC, Mean corpuscular hemoglobin concentration; MCV, Mean corpuscular volume; ND4-ND5, NADHdehydrogenase subunits 4 and 5; PLT, Platelet; RBC, Red blood cell; WBC, White blood cell