Doctoral Degrees (Molecular Biology and Human Genetics)
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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by Author "Clarke, Charlene"
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- ItemInvestigating host and pathogen biomarkers of mycobacterium bovis and nontuberculous mycobacterial infection in African buffaloes (Syncerus caffer)(Stellenbosch : Stellenbosch University, 2023-01) Clarke, Charlene; Miller, Michele Ann; Goosen, Wynand Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH 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.