Browsing by Author "Clarke, Charlene"

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    Animal-adapted members of the Mycobacterium tuberculosis complex endemic to the southern African subregion
    (AOSIS Publishing, 2016-04) Clarke, Charlene; Van Helden, Paul; Miller, Michele Ann; Parsons, Sven
    Members of the Mycobacterium tuberculosis complex (MTC) cause tuberculosis (TB) in both animals and humans. In this article, three animal-adapted MTC strains that are endemic to the southern African subregion – that is, Mycobacterium suricattae, Mycobacterium mungi, and the dassie bacillus – are reviewed with a focus on clinical and pathological presentations, geographic distribution, genotyping methods, diagnostic tools and evolution. Moreover, factors influencing the transmission and establishment of TB pathogens in novel host populations, including ecological, immunological and genetic factors of both the host and pathogen, are discussed. The risks associated with these infections are currently unknown and further studies will be required for greater understanding of this disease in the context of the southern African ecosystem.
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    Development and evaluation of a diagnostic cytokine-release assay for mycobacterium suricattae infection in meerkats (Suricata suricatta)
    (BioMed Central, 2017-01-04) Clarke, Charlene; Patterson, Stuart James; Drewe, Julian Ashley; Van Helden, Paul David; Miller, Michele Ann; Parsons, Sven David Charles
    Background: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. Results: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. Conclusion: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals.
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    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.
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    Investigation of temporal changes in immune responses to Mycobacterium bovis in cattle and African buffaloes (Syncerus caffer).
    (Stellenbosch : Stellenbosch University, 2017-03) Clarke, Charlene; Miller, Michele Ann; Parsons, Sven David Charles; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Cattle and African buffaloes are major maintenance hosts of Mycobacterium bovis in South Africa and therefore serve as a potential source of infection for other animals and humans. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB). Identification and removal of M. bovis-infected animals from the herd at early stages of infection is important for effective disease management and relies on the strategic application of ante-mortem diagnostic tests. Ante-mortem diagnostic tests rely on the detection of specific immunological responses in the animal. However, these responses vary over the course of infection and therefore the accuracy of the diagnostic test may change, depending on the stage of infection. We therefore aimed to investigate the temporal changes in antigen-specific cytokine production and humoral responses to BTB in buffaloes. Interferon-gamma (IFN-ɣ) and IFN-ɣ-induced protein 10 (IP-10) production in response to mycobacterial antigens ESAT-6/CFP-10, and serum antibodies to bovine purified protein derivative (bPPD) were measured in a group of chronically test-positive, newly converted test-positive and test-negative buffaloes over one year. No consistent trends in immune responses over time were observed in any of the groups. Cytokine release assays (CRAs) are often used in conjunction with the tuberculin skin test (TST) to improve diagnostic performance. However, many studies have shown that the TST may influence IFN-ɣ production and thereby the outcome of immunodiagnostic tests in cattle. However, these results were conflicting. A better understanding of the influence of the TST on cytokine production and the duration of this effect in a South African situation is required. We therefore aimed to investigate if the TST has an influence on IFN-ɣ and IP-10 production and test outcome in cattle and African buffaloes. IFN-ɣ and IP-10 release were measured in response to bPPD, avian PPD, ESAT-6 and CFP-10 in groups of TST-positive cattle 6, 7, 21, 41 and 78 days post-TST and in a group of TST-negative cattle 7, 21 and 78 days post-TST. IFN-ɣ and IP-10 levels in response to PPD were elevated 1-3 weeks post-TST, followed by a decrease by 41 days, suggesting immune boosting by the TST. ESAT-6/CFP-10-specific cytokine release showed conflicting results, with a group of animals showing decreased cytokine production by 41 days, whereas another group showed no change in cytokine release over time. Our findings suggest that cattle should not be tested with CRAs between 6 and 41 days post-TST to avoid boosting of cytokine levels and inaccurate test results. IFN-ɣ and IP-10 release in response to PC-EC®, PC-HP®, bPPD and avian PPD were measured in a group of Bovigam®-positive and negative buffaloes at the time the TST was performed and three days later. In Bovigam®-positive buffaloes a significant decrease in cytokine production and in the proportion of test positive animals were observed three days post-TST in response to all antigens, except aPPD. Bovigam®-negative animals were not influenced by the TST. It is therefore recommended that buffaloes should be sampled pre-TST to identify all possible M. bovis-positive animals in this herd of high BTB incidence. Accuracy of diagnostic assays may be affected by many factors, including the immunological stage of M. bovis-infection, time interval between the performance of the TST and CRA, exposure to environmental mycobacteria and BTB incidence in the herd. The correct application and interpretation of diagnostic assays of high specificity and sensitivity is required to overcome some of these factors. Therefore, information of the BTB status of the herd, exposure to environmental mycobacteria, and an understanding of the aim of the disease management plan is required.
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    Novel molecular transport medium used in combination with Xpert MTB/RIF ultra provides rapid detection of Mycobacterium bovis in African buffaloes
    (Nature Research (part of Springer Nature), 2021) Clarke, Charlene; Smith, Katrin; Goldswain, Samantha J.; Helm, Christopher; Cooper, David V.; Kerr, Tanya J.; Kleynhans, Leanie; Van Helden, Paul D.; Warren, Robin M.; Miller, Michele A.; Goosen, Wynand J.
    ENGLISH ABSTRACT: Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.
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    Review of diagnostic tests for detection of mycobacterium bovis infection in South African wildlife
    (Frontiers Media S.A, 2021-01) Bernitz, Netanya; Kerr, Tanya J.; Goosen, Wynand J.; Chileshe, Josephine; Higgitt, Roxanne L.; Roos, Eduard O.; Meiring, Christina; Gumbo, Rachiel; De Waal, Candice; Clarke, Charlene; Smith, Katrin; Goldswain, Samantha; Sylvester, Taschnica T.; Kleynhans, Léanie; Dippenaar, Anzaan; Buss, Peter E.; Cooper, David V.; Lyashchenko, Konstantin P.; Warren, Robin M.; Van Helden, Paul D.; Parsons, Sven D. C.; Miller, Michele A.
    Wildlife tuberculosis is a major economic and conservation concern globally. Bovine tuberculosis (bTB), caused byMycobacteriumbovis (M. bovis), is themost common form of wildlife tuberculosis. In South Africa, to date, M. bovis infection has been detected in 24 mammalian wildlife species. The identification of M. bovis infection in wildlife species is essential to limit the spread and to control the disease in these populations, sympatric wildlife species and neighboring livestock. The detection of M. bovis-infected individuals is challenging as only severely diseased animals show clinical disease manifestations and diagnostic tools to identify infection are limited. The emergence of novel reagents and technologies to identify M. bovis infection in wildlife species are instrumental in improving the diagnosis and control of bTB. This review provides an update on the diagnostic tools to detect M. bovis infection in South African wildlife but may be a useful guide for other wildlife species.