Molecular Identification of Trypanosoma theileri and Biology of Trypanosomes

Abstract

Trypanosoma theileri (T. theileri ) is a non-pathogenic, cosmopolitan, and commensal protozoa of cattle. The main objective of the current study was to investigate the biology and feasibility of T. theileri as a model candidate for the discovery of a novel drug. In the present study, the isolates of T. theileri obtained from the Institute of Tropical Medicine (ITM) in SDM 79 were cultivated at 26oC. Eight experiments with different inoculum and different times were grown. The growth curve was plotted to check the growth trends. The doubling time in the logarithmic phase was determined to be 17.43 hours. In addition, an experimental infection was done on a 3-month-old Holstein Friesian calf to isolate the blood-streaming shape; however, it was not successful after the blood buffy coat smear and PBMC culture in RPMI 1640 and HMI 9. Furthermore, the viability was determined by quantitative colorimetric Resazurin assay in 96-well fluorescence Microplates containing 0.4 to 2.4 mM of Resazurin. On the other hand, the response to Pentamidine (1-100 ng/mL) showed a strong negative correlation between the fluorescence signal and the highest Pentamidine concentration. IC50 was 9.25 ng/mL. Genomic DNA was extracted using the phenol-chloroform method. The gradient PCR amplification using T. theileri specific PCR (Tth625-PCR) primers was detected at 465 base pair (bp). In addition, the full-length 18S rDNA sequence was detected at 730 bp. In the silico analysis using common anti-trypanosome drug targets, no significant similarity could be found on either the DNA or the protein level. Nevertheless, homologous sequences have been identified among the drug targets for Ornithine decarboxylase. Therefore, the analysis might show the possibility of using T. theileri as a model for the search of new drugs once they have entire genome sequences. Analysis of the whole genome and transcriptome indicated a phylogenetic relationship between T. theileri and other pathogenic trypanosomes which can be the basis for novel drug development.

Microscopic study of developmental stages of a Trypanosoma theileri-like trypanosome in axenic culture

“…Not surprisingly, many works have been devoted to the cultivation of the parasites isolated from the blood of the vertebrate host as well as the use of such cultures in various functional studies (Wink, 1979;Townsend et al, 1982;Oyamada et al, 1995;Lee et al, 2013). Recently, T. theileri has been used as a model object for the development of trypanocidal drugs (Fentahun, 2020;Fentahun and Paeshuyse, 2021). At the same time, information on the ultrastructure of these parasites is very scarce and often devoted only to the issue of their interaction with cells in a tissue culture, on which they are grown (Townsend and Duffus, 1982;Griebel et al, 1989;Oyamada et al, 1995;Rodrigues et al, 2003;Lee et al, 2013).…”