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Mousa, Awadia Mohamed Ali Mohamed (2010): Identification of attenuation markers of a Theileria lestoquardi cell line to be used for the development of live vaccine against malignant ovine theileriosis. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Theileria lestoquardi is a tick-borne protozoan parasite and highly pathogenic for sheep. The disease caused by the pathogen is known as malignant ovine theileriosis (MOT) and is transmitted by Hyalomma ticks. Control of the disease can be achieved by immunization of sheep with attenuated T. lestoquardi schizont-infected ovine cells that provides the animal with solid immunity. The approach of using the attenuated vaccine against malignant ovine theileriosis has been carried out successfully in Iraq and Iran. Better characterization of attenuated cell lines could result in the identification of markers that would allow more rapid selection of attenuated vaccine and reduce the cost of vaccine production. Since no work has been reported regarding attenuation mechanisms in T. lestoquardi, the following study investigated potential attenuation markers of T. annulata infected cells in a T. lestoquardi cell line at different passages. Two markers associated with attenuation in T. annulata vaccine strains were analyzed, matrix metalloproteinase activity and TNF-alpha mRNA expression. Furthermore, differentially expressed genes in higher passage and lower passage were analyzed using suppression subtractive hybridization in order to identify genes whose expression correlates with subculturing and thus potentially with attenuation. The expression of matrix metalloproteinase-9 (MMP9) and matrix metalloproteinase-2 (MMP2) in the investigated cell line was confirmed by using specific inhibitors. The results showed gradual reduction in the activity of matrix metalloproteinase-9 (MMP9) with increasing passage number. Following the mRNA expression of TNF-alpha in different passages revealed down regulation of this cytokine from the low passage compared with high passage. Analysis of randomly selected clones in the suppression subtractive hybridization libraries identified nine differentially expressed genes, one from the parasite and eight from the host. Transcripts of retinoblastoma binding protein 7, Enolase-a (ENO 1), Ki-67 antigen and H2A histone from the host and vacuolar H+ATPase from the parasite were more plentiful in low passage culture. RAB14, a member of the RAS oncogene family, glucose transporter type 3, creatine kinase B, and cytochrome C oxidase transcripts from the host were more abundant in high passage culture. Quantitative real time-PCR confirmed mRNA expression of the parasite vacuolar H+ATPase to be downregulated at higher passages. The expression of the Ki-67 protein was clearly decreased with increasing passage number in western blot using specific antibody. Moreover, assessment of thymidine incorporation as a measure for the proliferation rate clearly showed that with increasing passage number, the proliferation rate of the T. lestoquardi infected cells decreases. This study revealed that the matrix metalloproteinase enzymes (9 and 2) and TNF-alpha could be potential molecular markers for identification of attenuation in the Theileria lestoquardi (Atbara) cell line. Also the down regulated parasite gene, vacuolar H+- ATPase could be considered as a molecular marker for attenuation. Immunization trials in sheep with different passages are required to provide in vivo evidence to support these findings.