Masters Degrees (Molecular Biology and Human Genetics)

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Browsing Masters Degrees (Molecular Biology and Human Genetics) by Subject "African elephant"

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    Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana)
    (Stellenbosch : Stellenbosch University, 2021-03) De Waal, Candice Raquel; Miller, Michele Ann; Kerr, Tanya Jane; Kleynhans, Leanie; Landolfi, Jennifer; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: African elephants (Loxodonta africana) are considered priority species within conservation areas because of their aesthetic value, ecological importance, and economic contribution to the ecotourism industry. Conservation efforts have focused on protecting habitat, but there are fewstudiesinvestigating the role of disease. The recent discovery of tuberculosis (TB) in a free-ranging African elephant in Kruger National Park (KNP), South Africa, has resulted in movement restrictions, preventing the translocation of elephants from this population. Since diagnostic tests for TB in wildlife are limited, the development of blood-based teststo detect Mycobacteriumtuberculosiscomplex (MTBC) infection in African elephants isneeded. These antigen-specific immune assays would have a significant beneficial impact on current practices in wildlife and zoological medicine. Therefore, the aim of this project was to identify blood-based host biomarkers that can be used to detect immune responses of African elephants. Cytokine gene expression assays (GEAs) have been employed to measure cell-mediated immune responses in a variety of species. These GEAs use real-time, reverse-transcription quantitative PCR (RT-qPCR) to measure changes in gene expression of immune cells,following stimulation ofwhole blood. In this study, wholeblood from African elephants from KNP, a Mycobacteriumbovis-endemic area,wasstimulated using pokeweed mitogen and mycobacterial antigens. Newlydesigned primers, as well as modified primers originally developed for useinother species, were used to amplifyand sequence African elephant mRNA transcripts of selected target(CXCL9, CXCL10, IFNγ, IL4, IL10, IL12, TGFβ,andTNF)and reference genes(ACTB, B2M, GAPDH, YWHAZ). These mRNA transcriptswere used to design sequencespecific primers and develop a RT-qPCR to determine changes in cytokine expression as a measure of general immune activation and antigen-specific responses. Confirmed mRNA transcriptsfor African elephants were used to develop real-time RT-qPCRs forIL10, TNF,andTGFβ,relative to GAPDHas the optimal reference gene.Thesecytokine GEAsdemonstrated the use of identified biomarkers to measure immune responses in this species. To our knowledge, this was the first study that has investigatedcytokine biomarkers in African elephants using real-time RT-qPCR. Results of the cytokine GEAs showed up-regulation of IL10and TNF, as well as down-regulation of TGFβ,in response to mitogen stimulation. When expression of these cytokines wasevaluated in response to mycobacterial antigen stimulation, asignificantup-regulation of IL10was observed following PPDa and PPDb stimulation. However, following stimulation with ESAT6/CFP10, as well as calculated differential PPD response, a very slight down-regulation of IL10was observed, as expected in TB uninfected elephants. Similarly, a slight down-regulation of TNFand TGFβwas observedfollowing all mycobacterial antigen stimulations. Findingsin this study provide novel insights into the African elephant immune system. The generated mRNA transcripts provide a basis for development of immunological assays for TB, as well asother diseases. Finally, evaluation ofgene expression following antigen stimulation provided insight into the use of PPDa, PPDb and ESAT6/CFP10 as stimulantsof antigen-specific TB responses. This will aid in the development of tools to improve disease detection and diagnosisin African elephants.