Identification of Novel Genes for the Development of a Rapid Diagnostic Test for Theileria uilenbergi Infection by Screening of a Merozoite cDNA Library

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.