Semi-quantitative Analysis of Expression of Various Genes in relation to Possible Markers for Theileria annulata Attenuation

The sporozoites of Theileria annulata invade bovine MHC II cells, where they differentiate into schizonts. The later can immortalize and induce fundamental changes in their host cells. Live attenuated vaccine is an important way of controlling T. annulata infection of cattle. Production is by prolonged cultivation of macroschizont-infected cells. The mechanisms underlying this transformation are not understood. The objects of this work were to analyze the expression levels of MMPs, Pro-inflammatory cytokines, Tams1 and TashHN genes in relation to possible markers for Theileria annulata attenuation. Semi-quantitative polymerase chain reaction (RT-PCR) was applied to quantify and compare variations in gene expression level among different passage numbers of three cell lines. The results of this study demonstrated that the infected cells show detectable specific transcripts for MMP9 in low passage cultures, but it decreased in long term passages (S15 vaccine strain and high passage number of C1 and C2 cell lines). The analyses of three available cell lines indicated detectable amount of specific mRNAs for TashHN. Tams1 specific transcripts were detected in low passage number of C1 and C2 cell lines, but not obtained in attenuated S15 vaccine and prolonged culture of C1 and C2 cell lines. Two pro-inflammatory cytokines, IL-1-beta and TNF-alpha, were detected with high fluctuations in all three T. annulata infected cell lines, both in low and high passage number. In conclusion, the results of this work clearly showed that the level of MMP9 transcripts is in contrast with the amounts of Tams1 mRNAs in T.annulata schizont infected cell lines that might be considered for virulence and attenuation respectively. Understanding the mechanisms of virulence and attenuation of infected cell line by using molecular biology methods and in vivo animal experiments could help to increase our knowledge about attenuation mechanisms and preparing and identifying appropriate cell lines in order to develop the new T. annulata vaccine cell lines.