Podcasts about theileria

Vincent, Dickson, and Daniel discuss how a secreted protein from the protozoan parasite Theileria transforms its host cells via a cellular proto-oncogene. Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Links for this episode: Leishmania (TWiP #14) Transformation by a prolyl isomerase (Nature) Theileria.org Prolyl isomerase (Wikipedia) Image from Transformation & Oncogenesis Letters read on TWiP 88 Contact Send your questions and comments (email or mp3 file) to twip@twiv.tv Subscribe Subscribe to TWiP (free) in iTunes, by the RSS feed or by email

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.

The major aim of this thesis was to identify novel genes of T. uilenbergi through establishment and screening of a merozoite cDNA library with the eventual goal to develop diagnostic tools using identified genes for detection of Theileria infections. The experiments were initiated by infection of sheep using T. uilenbergi stock. When parasiteamia rose, blood was collected and the merozoites were purified. Messenger RNA was isolated from purified merozoites was then utilized to establish a cDNA library. The library was titrated to be 6 x 108 pfu/ml and the recombinant clones were estimated to be 70%. Random PCR identification of the library indicated all of the inserts were of parasite origin, indicating the usefulness of the library for the identification of new genes. Random PCR amplification of inserts of the cDNA library led to the discovery of 12 single clones, among which Clone 2, 9 and 26 exhibited a high degree of identity, especially at the 3' terminus and 3' untranslated region, indicating that they belong to the same gene family. Furthermore, PCR designed to target Clone 2 amplified again four variant genes from genomic DNA of T. uilenbergi and one from genomic DNA of T. luwenshuni, suggesting this gene family is likely isolate-specific since the DNA samples for PCR were not derived from the same parasite isolate used for library construction. Sequence analysis of another genomic fragment generated with primers targeting the 3' untranslated region of the Clone 26 sequence showed that both 5' and 3' termini were highly identical to the Clone 2 gene family and these homologous termini were separated by a 136 bp sequence fragment highly identical to the 3' untranslated region of the Clone 2 gene family, indicating Clone 2 gene family members are tandemly arranged. Bioinformatic analysis of cDNA sequences of the Clone 2 gene family indicated these genes contain signal peptides and encode potential immunogenic proteins. Analysis of recombinantly expressed Clone 2 revealed immunoreactivity with sera from Theileria-infected animals from China. No cross reaction with sera of T. lestoquardi-, Babesia motasi- or Anaplasma ovis- infected animals was observed, indicating a potential specificity of this gene family. The features of the Clone 2 gene family are similar to the Tpr gene family of T. parva, which is believed to play a role in concerted evolution. Based on the highly conserved region of the Clone 2 gene family, a set of six primers were designed for the development of a loop mediated isothermal amplification (LAMP). The established assay allowed the detection of T. uilenbergi and T. luwenshuni infections simultaneously and the reaction could be simply accomplished by incubation at 63ºC for 15 min. The specificity of the reaction was confirmed through EcoRI restriction enzyme digestion analysis and sequencing. The assay was sensitive as it detected 0.1 pg DNA of T. luwenshuni or T. uilenbergi. Moreover, the assay was evaluated by testing 86 field samples in comparison to the reverse line blot method, showing a calculated sensitivity and specificity of 66.0% and 97.4%, respectively. These results indicate that the LAMP assay has a potential usefulness for application in diagnostic and pidemiological studies on T. luwenshuni and T. uilenbergi infection of small ruminants. In addition, serological screening of the library led to discovery of a positive clone called TuIP, which has been deposited in Genbank under accession number FJ467922. Partially recombinantly cloned and expressed TuIP showed strong reactivity with serum from T. uilenbergi infected animals, indicating its potential usefulness for development of novel serological diagnostic tests or serving as a candidate for vaccine development in the future.