Abstract:
The apicomplexan parasite Theileria annulata is transmitted by Hyalomma ticks and causes an acute
lymphoproliferative disease that is invariably lethal in exotic cattle breeds. The unique ability of the
schizont stage of T. annulata to transform infected leukocytes to a cancer-like phenotype and the
simplicity of culturing and passaging T. annulata-transformed cells in vitro have been explored for live
vaccine development by attenuating the transformed cells using lengthy serial propagation in vitro.
The empirical in vivo evaluation of attenuation required for each batch of long-term cultured cells
is a major constraint since it is resource intensive and raises ethical issues regarding animal welfare.
As yet, the molecular mechanisms underlying attenuation are not well understood. Characteristic
changes in gene expression brought about by attenuation are likely to aid in the identifcation of
novel biomarkers for attenuation. We set out to undertake a comparative transcriptome analysis
of attenuated (passage 296) and virulent (passage 26) bovine leukocytes infected with a Tunisian
strain of T. annulata termed Beja. RNA-seq was used to analyse gene expression profles and the
relative expression levels of selected genes were verifed by real-time quantitative PCR (RT-qPCR)
analysis. Among the 3538T. annulata genes analysed, 214 were signifcantly diferentially
expressed, of which 149 genes were up-regulated and 65 down-regulated. Functional annotation of
diferentially expressed T. annulata genes revealed four broad categories of metabolic pathways:
carbon metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum and
biosynthesis of secondary metabolites. It is interesting to note that of the top 40 genes that showed
altered expression, 13 were predicted to contain a signal peptide and/or at least one transmembrane
domain, suggesting possible involvement in host-parasite interaction. Of the 16,514 bovine
transcripts, 284 and 277 showed up-regulated and down-regulated expression, respectively. These
were assigned to functional categories relevant to cell surface, tissue morphogenesis and regulation
of cell adhesion, regulation of leucocyte, lymphocyte and cell activation. The genetic alterations
acquired during attenuation that we have catalogued herein, as well as the accompanying in silico
functional characterization, do not only improve understanding of the attenuation process, but can
also be exploited by studies aimed at identifying attenuation biomarkers across diferent cell lines
focusing on some host and parasite genes that have been highlighted in this study, such as bovine
genes (CD69, ZNF618, LPAR3, and APOL3) and parasite genes such as TA03875.
