Browsing by Author "Chileshe, Josephine"

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    Identification of immunological biomarkers for detection of Mycobacterium bovis infection in African rhinoceros
    (Stellenbosch : Stellenbosch University, 2021-03) Chileshe, Josephine; Miller, Michele Ann; Parsons, Sven D. C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: African rhinoceros have economic and ecological value, yet they are threatened by poaching and habitat loss, which causes reduction in population numbers. In addition, infection with Mycobacterium bovis(M. bovis), a causative agent of bovine tuberculosis (bTB), may be a threat to their conservation. This legislatively notifiable infectious disease has serious consequences on management of African rhinoceros by restricting their movements; therefore, it is imperative to have accurate and easily available diagnostic tools for the detection of M. bovisinfection, to ensure that infected rhinoceros are not translocated. In order to address this need, this study aimed to (i) optimize enzyme-linked immunosorbent assay (ELISA) conditions for detecting rhinocerosinterferon-gamma (IFN-γ), (ii) develop a diagnostic antigen-specific IFN-γ release assay (IGRA) for rhinoceros, (iii) identify cytokine gene signatures that distinguish between M. bovis-infected and uninfected rhinoceros, and (iv) identify cytokines other than IFN-γ with diagnostic potential to distinguish between M. bovis-infected and uninfected animals. Whole blood samples were collected from M. bovis-infected rhinoceros and uninfected animals from the Kruger National Park and incubated overnight at 37°C in tubes of the QuantiFERON (QFT) system. Thereafter, harvested plasma samples and cell pellets preserved in RNAlater, were stored at -80°C. Archived plasma was used to validate the equine Mabtech IFN-γ ELISAPROand then develop a diagnostic IGRA for rhinoceros.Using archived blood pellets, RNA was extracted, reverse transcribed, and screened using the equine RT2profiler PCR array to identify candidate cytokine biomarkers. In addition, the Raybio® equine interferon gamma induced protein (IP-10) ELISA was used with rhinoceros plasma from QFT stimulated blood to investigate detection of rhinoceros IP-10. The commercial Mabtech Equine IFN-γ ELISAPROkit was validated as a robust and highly reproducible assay for the detection of rhinoceros IFN-γ. Thereafter, the QFT IGRA was shown to distinguish between M. bovis-infected and uninfected animals. The optimal cut-off value was calculated as 21 pg/ml, although test results ranging between 22–61 pg/ml were interpreted as indeterminate, and a test result of >62 pg/ml as definitively positive.Using RNA extracted from QFT-stimulated rhinoceros whole blood, theequine RT2profiler PCR array identified six potential cytokine gene targets. Further development of a gene expression assay for CXCL10 (IP-10)confirmed this cytokine as a promising biomarker based on significant upregulation in antigen-stimulated blood from M. bovis-infected rhinoceros. Based on this result, an equine IP-10 release assay (IPRA) was developed to demonstrate that antigen-specific IP-10 could discriminate between M. bovis-infected and uninfectedwhite rhinoceros. Additionally, the IPRA was able to detect one additional truly infected animal when used in parallel with the IGRA, suggestingthat this may have potential as an additional diagnostic biomarker in rhinoceros.In summary, the novel tools developed in this project can be used to detect M. bovisinfection in rhinoceros. Similar to other species, QFT IGRA and potentially IPRA may support testing of individual rhinoceros prior to translocation, as well as allow for disease surveillance. Future research should focus on additional biomarker discovery and understanding the rhinoceros’ immune response to M. bovisinfection.
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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.