Doctoral Degrees (Molecular Biology and Human Genetics)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3556

Browse

Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by Subject "African buffalo"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Investigating 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.
  • Thumbnail Image
    Item
    Novel approaches to the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer)
    (Stellenbosch : Stellenbosch University, 2019-12) Bernitz, Netanya; Miller, Michele Anne; Parsons, Sven David Charles; Du Plessis, Nelita; Stellenbosch University. Faculty Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
    ENGLISH ABSTRACT: Mycobacterium bovis(M. bovis) is the pathogen that causes bovine tuberculosis (bTB) in a wide range of host species including livestock and wildlife. Globally, the control of M. bovisinfection is hindered by the existence of wildlife maintenance hosts. In South Africa, African buffaloes (Syncerus caffer) are considered maintenance hosts of bTB, and therefore control in this species will facilitate control in other sympatric wildlife species and livestock. With the limited availability of diagnostic tools and their suboptimal test performancesto detectM. bovisinfection in buffaloes, it is imperative to develop novel approaches to improve the detection ofinfected buffaloes. In this study, the QuantiFERON®TB-Gold (QFT) system in combination with the cattletype®IFN-gamma ELISA, the QFT interferon gamma (IFN-γ)release assay (IGRA), was shown to have high specificity but poor sensitivity in detecting M. bovisinfection in buffaloes. The sensitivityof the QFT IGRA was improved by measuring the chemokineIFN-γ-inducible protein-10 (IP-10)in theQFTIP-10release assay (IPRA). When both cytokines IFN-γ and IP-10 were measured in parallel in the QFT system, sensitivity was further improved and the specificityof the individual assays were maintained. The concentrations of IFN-γ and IP-10 in QFT tubes were used to predictthe presence of macroscopic pathology in M. bovis-infected buffaloes. Lastly, the immunophenotyping of cattle whole blood identified cellular subsets of bovine leukocytes,however, the production of IP-10 in these cells wasnot confirmed. This study has demonstrated that the QFT system is a highly practical stimulation platform to detect M. bovisinfection in buffaloes with high specificity. The QFT system and novel cattletype®IFN-gammaELISA is anIGRA with high specificity that can be used to detect M. bovisinfection in buffalo populations. The cytokine IP-10 is a more sensitive biomarker than IFN-γ and when these two cytokines are measured in parallel in the QFT system, the detection of infected buffaloes is maximised, the specificityis high and the testing procedure is simplified. Finally, the magnitude of IP-10 and IFN-γ concentrations in QFT-processed whole blood can be usedas indicators of bTB pathology in M. bovis-infected buffaloes