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Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana)
Thesis (MSc)--Stellenbosch University, 2021.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867613922907914240 |
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| access_status_str | Open Access |
| author | De Waal, Candice Raquel |
| author2 | Miller, Michele Ann |
| author_browse | De Waal, Candice Raquel Miller, Michele Ann |
| author_facet | Miller, Michele Ann De Waal, Candice Raquel |
| author_sort | De Waal, Candice Raquel |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/109939 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:43:50.825Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/109939 Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) 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. African elephant Gene expression RT-qPCR Cell-mediated immunity UCTD Thesis (MSc)--Stellenbosch University, 2021. 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. AFRIKAANSE OPSOMMING: Afrika-olifante (Loxodonta africana) word as ʼn prioriteitspesies in bewaringsgebiedebeskou, vanweë hul estetiese waarde, ekologiese belang en ekonomiese bydrae tot ekotoerismebedrywighede. Bewaringspogings fokus grotendeels op die beskermingvan habitat, maar daar is 'n slegs n paar studieswat die rol van siektes in hierdie spesie ondersoek. Die onlangse ontdekking van tuberkulose (TB) in 'n vrylopende Afrika-olifant in die Krugerwildtuin (KNP), Suid-Afrika, het gelei tot bewegingsbeperkingswat die skuif van Afrika-olifante uit hierdie bevolking verhinder. Aangesien diagnostiese toetse vir TB in wildsoorte beperk is, is die ontwikkeling van 'n bloedtoets om infeksie met Mycobacterium tuberculosiskompleks (MTBC) in Afrika-olifante op te spoor nodig.Antigeen-spesifieke immuuntoetse sal 'n voordelige impak op die bestaande praktyke in wild-en dierkundige medisyne hê. Die doel van hierdie projek was dus om bloedgebaseerde gasheer-biomerkers te identifiseer wat gebruik kan word om immuunresponse van Afrika-olifante te meet. Sitokien geenuitdrukkings-toetse (GEA’s) is voorheen gebruik om sel-gemedieerde immuunresponse in 'n verskeidenheid spesies te meet. Hierdie toetse gebruik ware-tyd, omgekeerde-transkripsie kwantitatiewe polimerase kettingreaksie (RT-qPCR) om veranderinge in geenuitdrukking in immuunselle, in gestimuleerde volbloedte meet.In hierdie studie is volbloed van Afrika-olifante vanuit KNP, ‘n Mycobacterium bovis endemiese area, deur middel van ‘n ‘pokeweed’ mitogeen asook mikobakteriële antigene gestimuleer. Nuut ontwerpte inleiers, asook gemodifiseerde inleierswat oorspronklik vir ander spesies ontwerp was, is gebruik om mRNA-transkripte van geselekteerde teiken-(CXCL9, CXCL10, IFNγ, IL4, IL10, IL12, TGFβ andTNF) en verwysingsgene (ACTB, B2M, GAPDH, YWHAZ) van Afrika-olifant te amplifiseer en die volgorder te bepaal. Hierdie mRNA-transkripte was toe gebruik om spesifieke sitokien inleiers teontwerp en 'n RT-qPCR te ontwikkel om veranderinge in sitokienuitdrukking te bepaal om sodoende algemene immuunaktivering en antigeenspesifieke reaksies te meet. Bevestigde mRNA-transkripte vir Afrika-olifante is gebruik om ware-tyd RT-qPCR vir IL10, TNFen TGFβte ontwikkel, relatief tot GAPDH as die optimale verwysingsgeen. Hierdie sitokien GEAs dui op die moontlike gebruik van die geïdentifiseerde biomerkers om immuunresponse in hierdie spesie te meet. Na ons wete was dit die eerste studie wat sitokien-biomerkers in Afrika-olifante ondersoek het deur gebruik te maak van ware-tyd RT-qPCR. Resultate van die sitokien-GEA's het ʼn toename in die IL10en TNFgeenuidrukking asook ʼn afnameing in TGFβgeenuitdrukking getoon nadat volbloed met mitogeen gestimuleer was. Toe die uitdrukking van hierdie sitokiene na mikobakteriële antigeenstimulasieevalueer was, het die uitdrukking van IL10beduident toegeneem na PPDa-en PPDb-stimulasie. Na stimulasie met ESAT6/CFP10, sowel as berekende differensiële PPD-response, is afwaartse regulering van IL10 egter waargeneem, in onbesmette olifante. Net so is afname inTNF en TGFβgeenuitdrukking waargeneem na aanleiding van mikobakteriële antigeensimulasies.Bevindings in hierdie studie bied nuwe insigte tot die immuunsisteem van die Afrika-olifant. Die gegenereerde mRNA-transkripte bied 'n basis vir die ontwikkeling van immunologiese toetse, nie net vir TB nie, maar ook vir ander siektes. Laastens bied hierdie ondersoek in geenuitdrukking na antigeenstimulasie insig oor die gebruik van PPDa, PPDb en ESAT6/CFP10 as aanduiders van antigeenspesifieke TB-reaksies. Dit sal help met die ontwikkeling van instrumente om die opsporing en diagnose van siektesin Afrika-olifantete verbeter. Masters 2021-02-08T11:10:44Z 2021-04-21T14:32:57Z 2021-02-08T11:10:44Z 2021-04-21T14:32:57Z 2021-03 Thesis http://hdl.handle.net/10019.1/109939 en_ZA Stellenbosch University 91 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | African elephant Gene expression RT-qPCR Cell-mediated immunity UCTD De Waal, Candice Raquel Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) |
| title | Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) |
| title_full | Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) |
| title_fullStr | Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) |
| title_full_unstemmed | Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) |
| title_short | Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana) |
| title_sort | characterization of biomarkers of immunological activation in african elephants loxodonta africana |
| topic | African elephant Gene expression RT-qPCR Cell-mediated immunity UCTD |
| url | http://hdl.handle.net/10019.1/109939 |
| work_keys_str_mv | AT dewaalcandiceraquel characterizationofbiomarkersofimmunologicalactivationinafricanelephantsloxodontaafricana |