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Liu, Zhijie (2010): Identification of Novel Genes for the Development of a Rapid Diagnostic Test for Theileria uilenbergi Infection by Screening of a Merozoite cDNA Library. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

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.