Development of a Competitive ELISA for Detection of<i>Theileria annulata</i>Infection

Renneker, Stefanie; Kullmann, Birgit; Gerber, Silke; Dobschanski, J.; Bakheit, Mohammed A.; Geysen, Dirk; Shiels, Brian; Tait, Andy; Ahmed, Jabbar S.; Seitzer, Ulrike

doi:10.1111/j.1865-1682.2008.01036.x

Cited by 23 publications

(14 citation statements)

References 16 publications

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“…However, issues such as low levels of parasite DNA in carrier animals [ 14 ] and competition between primers for species-specific conserved regions of certain genes such as 18S ssrRNA [ 16 , 17 ] serve to reduce test sensitivity: thus herd infection rates may be underestimated [ 16 , 18 , 19 ]. Additionally, for large-scale field studies molecular methods have the disadvantages of being expensive, labour intensive and require a degree of technical expertise [ 9 , 10 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

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Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata

et al. 2016

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Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals.

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Section: Introductionmentioning

confidence: 99%

“…Serological tests remain the most appropriate assays for large-scale studies aimed at identifying carrier animals and determining the distribution of the disease [ 20 , 21 ]. These rely on the fact that the bovine immune system is exposed to a variety of T .…”

Section: Introductionmentioning

confidence: 99%

Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata

et al. 2016

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“…No cross-reactions were found using the TaSp recombinant protein in ELISA analyzing anti- Babesia sera or sera from animals experimentally infected with Theileria mutans and Theileria parva . [ 11 , 12 ]. No cross-reactivity was recorded when Ta-LFD test was performed examining sera from animals experimentally infected with Babesia bovis, Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, Theileria mutans, and Theileria parva as well as serum from an animal testing positive in an ELISA for detection of infection with Theileria lestoquardi .…”

Section: Resultsmentioning

confidence: 99%

Assessment of the First Commercial ELISA Kit for the Diagnosis ofTheileria annulata

AL-Hosary

Ahmed

Nordengrahn

et al. 2015

Journal of Parasitology Research

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The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulata-Ab is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals.

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“…The schizont surface protein TaSP was detected by mouse monoclonal anti-TaSP (1C7 (Renneker et al, 2008;Shiels, McDougall, Tait, & Brown, 1986), rabbit polyclonal anti-TaSP (Schneider, Haller, Seitzer, Beyer, & Ahmed, 2004;Schnittger et al, 2002) or AlexaFluor488-labelled anti-TaSP antibodies, respectively. Further antibodies for immunofluorescence were mouse monoclonal antibodies anti-alpha-tubulin (clone DM1A, Santa Cruz Biotechnology), anti-CDK1 (clone POH-1, Santa Cruz), anti-phospho-threonineproline (Cell Signaling Technologies), anti-Ki67 (MIB-5; Dako) and rabbit polyclonal antibodies anti-CDK1 (clone H-297, Santa Cruz) and anti-TaHSP70.…”

Section: Antibody-based Immunofluorescence Staining and Confocal MImentioning

confidence: 99%

Theileria annulatasurface protein (TaSP) is a target of cyclin‐dependent kinase 1 phosphorylation in Theileria annulata‐infected cells

Mackiewicz

Seitzer

Ahmed

et al. 2020

Transbound Emerg Dis

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Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour‐like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite‐dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T131PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA‐based experiments using a phospho‐threonine‐proline (pThr‐Pro) antibody revealed, that CDK1‐cyclin B specifically phosphorylates T131, identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata‐infected cells. Further studies demonstrated a nearly complete co‐localization of the pThr‐Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle‐dependent event. Immunostainings of isolated, non‐permeabilized schizonts confirmed the presence of the pThr‐Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr‐Pro signal of the schizont, which was reconstituted by the addition of CDK1‐cyclin B. Treatment of T. annulata‐infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin‐rich cell protrusions and an extension of the schizont, and a dose‐dependent reduction of BrdU incorporation and Ki67 staining of T. annulata‐infected cells, demonstrating a clear impact on the Theileria‐dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria‐infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

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Development of a Competitive ELISA for Detection ofTheileria annulataInfection

Cited by 23 publications

References 16 publications

Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata

Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata

Assessment of the First Commercial ELISA Kit for the Diagnosis ofTheileria annulata

Theileria annulatasurface protein (TaSP) is a target of cyclin‐dependent kinase 1 phosphorylation in Theileria annulata‐infected cells

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