Fachbereich Veterinärmedizin



    Identification and characterization of a Theileria lestoquardi immunogenic protein and the application in a diagnostic ELISA (2004)

    Bakheit, Ahmed Mohammed Ahmed
    Berlin: Köster, 2004 — 107 Seiten
    ISBN: 3-89574-549-9
    Institut für Veterinär-Physiologie

    Oertzenweg 19 b
    14163 Berlin
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    Abstract / Zusammenfassung

    The main objectives of this study were to identify and characterize novel genes encoding for antigenic proteins of T. lestoquardi and to apply the recombinant proteins encoded by these genes in tools to diagnose malignant theileriosis of sheep and goats. The T. lestoquardi (Atbara) isolate originating from northern Sudan represented the main biological material used during this work. As this isolate was new, being isolated during this work, a first step was to characterize the isolate as T. lestoquardi. Thus, PCR experiments performed during the work confirmed absence of contaminating Theileria parasites, e.g. T. annulate, which could possibly exist in field isolates of T. lestoquardi. As a first step in the identification of new T. lestoquardi genes, a schizont cDNA library was established from partially purified schizonts of T. lestoquardi (Atbara). Isolated schizonts' integrity and the quality of the isolated mRNA were tested by immunostaining and PCR, respectively. The cDNA library was established using the SMART (Switching Mechanism At 5' end of RNA Transcript) cDNA synthesis technology and a A phage vector, which was capable of the simultaneous translation of all the three reading frames. The percentage of the white recombinant clones in the library was estimated to be 84.5%. It had a high titer of 6.2 x 109 pfu/ml and the percent of clones originating from T. lestoquardi (Atbara) schizont stage was tentatively estimated to be 80%. Immunoscreening of the library was carried out using sera collected from sheep recovered from natural malignant theileriosis. Several clones were selected, purified and sequenced either at random or on the basis of the immunoreactivity. One of these clones, against which the sheep sera samples were noticeably reactive, was chosen for further characterization and was given the name Clone-5. Characterization of Clone-5 gene and its protein forms was carried out using molecular biological experiments or predictions based on published bioinformatic methods. An interesting finding was the simultaneous presence of two transcript forms of the gene which implied the possibility of alternative splicing to occur in Theileria parasites. Thus two full length forms of the protein, a long form and a short form, were shown to occur in the schizont stage of the parasite, and their simultaneous existence was deduced by Western blot experiments. On one hand, Clone-5 gene showed most of the characteristics reported for genes encoding surface immunogenic proteins of apicomplexan parasites e.g. the presence of a leader sequence indicating a secretory pathway, the presence of sequence repeat motifs and the presence of immunogenic determinants. Immunocytological staining carried out using a rabbit polyclonal antiserum raised against one of the protein's antigenic determinants suggested the possible localization of at least one of the protein forms, presumably the long form, on the surface of T. lestoquardi schizonts. On the other hand, the sequence of the gene featured 13 protein kinase C (PKC) and 37 casein kinase II (CKII) phosphorylation sites, multiple nuclear localization signals (NLS) and a high probability (82.7%) of a nuclear sub-cellular localization. It could be hypothesized that the short form, being also a secretory protein but lacking transmembranal domains or GPI modification sites, may not be localized to the surface of the schizont, and as it contained NLS could target the host cell nucleus. Although the transfection experiments conducted to localize the protein within the subcellular compartments of the CHO cells were not conclusive, they suggested however, the presence of a functional cleavable leader sequence and the possibility of an extracellular transportation of the protein. The antigenicity of Clone-5 proteins was repeatedly confirmed during this study. An ELISA to detect antibodies against T. lestoquardi in the sera of infected animals was established using a recombinantly produced polypeptide derived from the sequence of Clone-5. A cut off value of 9% was determined using known negative sera samples. Preliminary tests carried out on a limited number of field sera (104 samples) and the counter-testing of the samples in IFAT revealed a correlation between the two tests in their judgment. However, there was no correlation noticed between the IFAT titers and the ELISA percent positivity values. Related to the IFAT, the ELISA had a sensitivity of 94.6% and a specificity of 88%. The results of this study show that Clone-5 ELISA could be a suitable tool for the diagnosis of malignant theileriosis sheep and goats and for testing larger numbers of sera in epidemiological surveys aiming at mapping out the distribution of this disease as part of malignant theileriosis control programs.