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 culture-adapted 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.

Fig. 1

The swelling of the genital organ of the dourine-infected stallion and sampling of trypanosomes from the urethral tract. a and b The swelling of the genital organ of the dourine-infected stallion. c HMI-9 was injected into the urethral tract using a transfer pipette to detach adherent T. equiperdum from the urethral tract mucosa. d Sampling of T. equiperdum from the urethral tract mucosa using a cotton swab

Fig. 2

Giemsa-stained T. equiperdum isolated from the urethral tract of the dourine-infected stallion. a and b (1K1N) show the single form of T. equiperdum. c (2K1N) and d (2K2N) show the dividing form of T. equiperdum. Arrow: nucleus; arrowhead: kinetoplast

Fig. 3

The PCR to detect NADH-dehydrogenase subunits 4 and 5 (ND4-ND5). A gel electrophoresis image of the PCR products of NADH-dehydrogenase subunits 4 and 5 (ND4-ND5). The arrowhead at 1515 bp indicates the amplicon of ND4-ND5 in lanes 1 and 3 to 7. M: 100 bp and 1 kbp DNA ladders, Lanes 1 to 8 are T. b. brucei GUTat3.1, T. evansi IL3960, T. equiperdum IVM-t1, STIB818, STIB841, STIB842, BoTat1.1 strains and a negative control (distilled water), respectively

Fig. 4

Akinetoplastic and kinetoplastic T. equiperdum in wild-type and in vitro culture. a T. equiperdum isolated from the urethral tract. Upper panel, akinetoplastic form; middle and lower panels, kinetoplastic form. b Kinetoplastic and akinetoplastic T. equiperdum in vitro culture. Arrow: nucleus; arrowhead: kinetoplast