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. 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. 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. 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.

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.

Ambient heat exposure reduces male fertility in mammals with scrotal testes. Our previous work has demonstrated that some stallions are more susceptible to ambient heat-related subfertility than others, yet the mechanism for heat-induced subfertility remains uncertain, limiting both diagnosis and preventative measures. This study sought to define how the phenotype of stallions susceptible to heat-induced subfertility differs from that of more resilient animals, by measuring the systemic (blood plasma) and localized (reproductive tract) inflammatory and oxidative stress markers of sperm concentration, sperm motility assessments, total antioxidant capacity (TAC; in blood and seminal plasma), malondialdehyde (MDA; in blood and seminal plasma), oxidized guanine species (8-OH-2dG; in blood plasma and spermatozoa DNA), sperm DNA damage (assessed via Halo, SCSA (Sperm Chromatin Structure Assay) and CMA3 (Chromomycin A3)), and c-reactive protein (CRP; in blood plasma). Post-breeding dismount semen samples (n = 357) and blood plasma samples (n = 97) were collected from 31 stallions at commercial thoroughbred studs throughout one breeding season (NSW, Australia). A subset of stallions (16%) was deemed heat-induced subfertility-susceptible (HISS) stallions. These animals showed reduced seminal plasma antioxidant capacity, increased systemic and localized lipid peroxidation, and distinct systemic inflammatory response. Seminal antioxidant capacity was found to be strongly associated with impaired sperm motility (r = 0.739 * vs. r = −0.059). The plasma c-reactive protein of heat-susceptible stallions correlated to heat exposure (r = 0.597 *) and affected sperm motilities (r = −0.527 **, r = −0.434 *). Systemic oxidative DNA damage (8-OH-2dG) also increased following heat events (r = 0.862 ***) and correlated with fertility losses (FCP: r = −0.740 **, PCP: r = −0.603 *). Non-HISS stallions displayed greater variability in systemic antioxidant status and robust response following heat exposure (r = 0.307 *) and localized antioxidant capacity was more strongly correlated to systemic antioxidant capacity than in the heat-susceptible group (r = 0.897 *** vs. r = 0.482 **). We demonstrate that impaired antioxidant responses, altered redox balance and suppressed acute-phase inflammatory signalling are key features associated with heat-induced subfertility in stallions and highlight biomarkers that could be used to identify animals with heat-susceptible fertility.