Reduction of Trypanosoma equiperdum from equine semen by single layer centrifugation

Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 10(2) to > 5 x 10(6) trypanosomes, were added to semen samples. Subsequently, SLC was performed following standard procedures. The presence of the parasite in the purified semen was checked by wet smear examination, ITS1 PCR and in vivo inoculation in mice. Before SLC, all spiked semen samples, except the negative controls, were positive on PCR analysis. After SLC, all the pellets were found to be negative for T. equiperdum on microscopic examinations. Examination of the pellet by PCR could also not detect any parasite-DNA in the SLC-pellet of semen spiked with the lower number of parasites (10(2) to 10(4) trypanosomes). However, in the SLC pellets spiked with 10(4) - 5 x 10(4) trypanosomes, only 1 out of the 4 replicates was negative for parasite DNA. All groups spiked with > 5 x 10(4) trypanosomes were found to be positive on PCR. All mice in the positive controls exhibited parasitaemia (5/5). Mice inoculated with SLC-purified semen that was spiked with lower than 5 x 10(4) trypanosomes, remained free of parasitaemia, similar to the negative controls. However inoculation with SLC-pellets from samples with a higher number of trypanosomes (> 5 x 10(4) - 5 x 10(6) and > 5 x 10(6)), induced parasitaemia in 2 out of 5 and 3 out of 5 mice, respectively. This study indicates that single layer centrifugation can be used to clear T. equiperdum infected semen but that the success is dependent on the number of parasites.