Investigating host and pathogen biomarkers of mycobacterium bovis and nontuberculous mycobacterial infection in African buffaloes (Syncerus caffer)

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dc.contributor.advisorMiller, Michele Ann
dc.contributor.advisorGoosen, Wynand Johan
dc.contributor.authorClarke, Charlene
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2023-01-11T15:47:39Z
dc.date.accessioned2023-05-18T06:55:57Z
dc.date.available2023-01-11T15:47:39Z
dc.date.available2023-05-18T06:55:57Z
dc.date.issued2023-01en_ZA
dc.descriptionThesis (PhD)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH ABSTRACT: African buffaloes (Syncerus caffer) are important maintenance hosts of bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis). Accurate and rapid diagnoses are essential for detection of M. bovis infected buffaloes. However, various factors may impede diagnosis, including suboptimal sensitivity of mycobacterial culture and cross-reactive immune responses to nontuberculous mycobacteria (NTM). In addition, there are challenges with transporting samples from remote locations to laboratories, as well as risk of zoonotic infection of humans handling samples. This study broadly aimed to improve bTB diagnosis in buffaloes by investigating host and pathogen biomarkers of M. bovis and NTM infections. This was achieved by 1) evaluating a safe, rapid test procedure to detect the presence of pathogenic mycobacterial DNA in buffalo post-mortem and ante-mortem samples, 2) characterising NTMs present in buffaloes, and 3) developing a high specificity test algorithm to improve screening of historically bTB-free buffalo herds. PrimeStore® Molecular Transport Media (PS-MTM) ensures a safer bTB testing platform by inactivation of pathogens. In this study, it effectively preserved mycobacterial DNA in M. bovis infected buffalo oronasal and tissue swabs for molecular testing. The novel use of Xpert MTB/RIF Ultra with PS-MTM stored swab samples demonstrated that this commercial qPCR assay has promise for rapid, sensitive, and potentially in-field application, for detecting M. bovis shedding ante-mortem and infection post-mortem. A high diversity of NTM species was identified in a large percentage of buffalo oronasal secretion and tissue sample cultures by hsp65 or rpoB PCR amplicon Sanger sequencing, with M. avium complex members the most frequently detected. Genes encoding virulence factors, ESAT-6 and CFP-10, were present in more than half of the samples, which may be indicative of potential crossreactivity with bTB immunoassays. A commercial line-probe assay detected NTMs from DNA directly extracted from oronasal swabs, and the Ultra showed high specificity amidst the high presence of NTMs following direct application on oronasal swabs. Serial bTB test algorithms, with QuantiFERON® -TB Gold Plus (QFT) interferon-gamma (IFN-γ) release assays (IGRA) and QFT IFN-γ inducible protein-10 release assays (IPRA), showed the greatest specificity in a historically bTB-free buffalo herd, compared to parallel testing or individual tests. Presence of NTMs in oronasal swabs did not appear to impede assay specificity. In an unrelated historically bTB-free herd, serial testing accurately identified truly infected buffaloes (positive on IGRA and IPRA), which were confirmed M. bovis infected. In summary, buffaloes have a high diversity of NTMs present that may impede bTB diagnostic tests. However, assay specificity did not appear to be affected by NTM presence. High specificity was achieved when IGRA and IPRA were interpreted in series, and results demonstrated the value of this test algorithm to screen buffalo herds with no history of bTB. The Ultra assay maintained specificity in the presence of NTMs and accurately and rapidly identified M. bovis infected buffaloes post-mortem and ante-mortem from swabs stored in a pathogen inactivation media, PSMTM. These results demonstrate that rapid accurate differentiation of M. bovis infected and uninfected buffaloes can be achieved using methods described in this thesis. en_ZA
dc.description.abstractAFRIKAANS OPSOMMING: Afrika buffels (Syncerus caffer) is belangrike instandhoudingsgashere van bees tuberkulose (bTB), wat deur Mycobacterium bovis (M. bovis) veroorsaak word. Akkurate en vinnige diagnoses is noodsaaklik vir die verwydering van M. bovis-geïnfekteerde buffels. Verskeie faktore mag diagnose belemmer, soos sub-optimale kultuur sensitiwiteit en immunologiese kruis-reaksies tot nietuberkulêre mykobakterieë (NTM). Addisionele uitdagings sluit die vervoer van monsters vanaf afgeleë plekke na laboratoriums in, asook die risiko van opportunistiese soönotiese infeksies van diegene wat monsters hanteer. Die doelwit van hierdie studie was om bTB diagnose in buffels te optimiseer deur gasheer en patogeen biomerkers van M. bovis en NTM infeksies te bestudeer. Dit was bereik deur 1) ‘n veilige, vinnige toets prosedure te evalueer om patogeniese mykobakteriese DNS in post-mortem en ante-mortem buffel monsters op te spoor, 2) die karakterisering van alle NTMs teenwoordig in buffel monsters, en 3) die ontwikkeling van ‘n hoogs spesifieke toets algoritme om sifting van histories bTB-vrye kuddes te verbeter. PrimeStore® Molekulêre Transport Medium (PS-MTM) verseker ‘n veiliger bTB toets platform deur die inaktivering van patogene. In hierdie studie het dit effektief mykobateriese DNS in M. bovisgeïnfekteerde buffel oronasale- en weefsel deppers gepreserveer vir verdere molekulêre toetsing. Die gebruik van Xpert MTB/RIF Ultra met PS-MTM deppers demonstreer dat hierdie qPCR toets belowend is vir veilige, vinnige, sensitiewe, en potensieel in-veld toepassing, vir die opsporing van M. bovis vergieting en infeksie. Hoë NTM diversiteit was geïdentifiseer in ‘n groot persentasie buffel oronasale afskeidings en weefselmonster kulture deur hsp65 of rpoB PCR amplifisering en Sanger sekwensering. Gene wat esat-6 en cfp-10 kodeer, was teenwoordig in meer as die helfde van die monsters, wat mag aandui op moontlike kruis-reaktiwiteit met bTB immuuntoetse. ‘n Kommersiële “line-probe” toets kon NTMs direk vanaf geëkstaeerde DNS uit oronasale deppers opspoor, en die Ultra het hoë spesifisiteit getoon in die teenwoordigheid van NTMs na direkte toepassing op oronasale deppers. In vergelyking met individuele of parallelle toepassing van bTB toetse, het toets algoritmes in series, met QuantiFERON® -TB Gold Plus (QFT) interferon-gamma (IFN-γ) vrystellings toetse (IGRA) en QFT IFN-γ geïnduseerde protein-10 vrystellings toetse (IPRA), die grootste spesifisiteit in histories bTB-vrye buffel kuddes getoon. Die teenwoordigheid van NTMs in oronasale deppers het nie toets spesifisiteit beïnvloed nie. In ‘n onverwante histories bTB-vrye kudde, het toetse in series akkuraat positiewe buffels geïdentifiseer (positief op IGRA en IPRA), wat later bevestig was om M. bovis-geïnfekteer te wees. Ten slotte, al het buffels ‘n hoë diversiteit van NTMs teenwoordig, blyk dit egter dat toets spesifisiteit nie deur die teenwoordigheid van NTMs beïnvloed was nie. ‘n Hoë spesifisiteit was verkry toe IGRA en IPRA in serie geïnterpreteer was. Die resultate toon die waarde van hierdie toets algoritme om buffel kuddes wat histories bTB-vry is, te sif. Verder, die Ultra toets het spesifisiteit in die teenwoordigheid van NTMs onderhou, en kon vinnig en akkuraat M. bovisgeïnfekteerde buffels post-mortem en ante-mortem vanaf PS-MTM deppers identifiseer. Hierdie resultate demonstreer dat vinnige en akkurate differensiasie van M. bovis-geïnfekteerde en niegeïnfekteerde buffels bereik kan word deur die metodes wat in hierdie tesis beskyf word, te gebruik. af_ZA
dc.description.versionDoctoralen_ZA
dc.format.extent150 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126922en_ZA
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMycobacterium bovis en_ZA
dc.subject.lcshBiochemical markers en_ZA
dc.subject.lcshAfrican buffalo en_ZA
dc.subject.lcshBovine tuberculosisen_ZA
dc.titleInvestigating host and pathogen biomarkers of mycobacterium bovis and nontuberculous mycobacterial infection in African buffaloes (Syncerus caffer)en_ZA
dc.typeThesisen_ZA
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Doctoral Degrees (Molecular Biology and Human Genetics)