Identification of attenuation markers of a Theileria lestoquardi cell line to be used for the development of live vaccine against malignant ovine theileriosis

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.