Molecular Biology and Human Genetics

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Browsing Molecular Biology and Human Genetics by Subject "African buffalo"

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    Characterisation of lesions and associated immune cell populations in the lung of African buffalo (Syncerus cafe) infected with Mycobacterium Boris
    (Stellenbosch : Stellenbosch University, 2021-03) Goldswain, Samantha; Miller, Michele; Kleynhans, Leanie; Jennifer, Landolfi; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: A vast array of species is susceptible toMycobacterium bovis (M. bovis) infection, with varying pathogenesis and disease outcomes. Understanding the pathogenesis of disease is important for informing epidemiologic concerns and disease management strategies, especially in reservoir hosts, such as African buffaloes (Syncerus caffer). Therefore, this project is focused on characterisation of histologic morphology and associated immune cell populations in lung granulomas from M. bovis infected African buffalo to advance knowledge of disease development in buffaloes. A scoring system was developed to compare numbers and distributions of different immune cells, as well as other pathologic changes in bovine tuberculosis (bTB) pulmonary granulomas. In addition, an immunohistochemistry (IHC) staining technique was optimised for immune cell surface marker detection in buffalo lung tissues. Formalin-fixed and frozen tissues were available from M. bovis naturally infected buffaloes from Hluhluwe-iMfolozi Park, South Africa. Formalin-fixed lung tissues were selected based on gross lesion scores that represented a range of severity. Lung sections from 14 buffaloes were stained with haematoxylin and eosin (H&E) to assess the histologic morphology of granulomas. Microscopic characteristics were then scored based on six categories. In addition, immunohistochemical techniques were optimised using antibodies that detected immune cell surface antigens (CD3, CD4, CD21, CD163, NCR1). This study documented characteristics of a low histologic stage granuloma to include macrophages/multinucleated giant cells (MNGCs) at the core, lymphocytic infiltration surrounding the macrophages, and minimal to no necrosis and fibrosis. Granulomas compatible with more advanced disease were necrotic, with macrophages surrounding the necrotic core, lymphocytes located peripherally, and were encapsulated by a fibrous capsule. More developed granulomas were not always mineralised. Based on IHC, CD3+ T lymphocytes and CD163+ macrophages/MNGCs were present in all granulomas examined; B cells (CD21+) were only present in higher stage granulomas; and natural killer (NCR1+) cells were not abundant in any of granulomas. The CD4 antibody did not stain buffalo tissues and therefore, distribution of this subset of T lymphocytes could not be evaluated. Ziehl-Neelsen staining was performed to detect the presence of acid-fast bacilli, however, no bacilli were visible in the slides analysed. The appearance of the buffalo pulmonary granulomas did not completely fit with the stages described in cattle (Wangoo et al., 2005). Therefore, a scoring system for categorising granulomas, adapted for buffaloes, should be investigated to provide a species-specific description that would be beneficial for understanding bTB pathogenesis. In summary, findings showed that lymphocytes and macrophages/MNGCs appear to be the predominant immune cell types present and their distribution and relative numbers appear to change as pulmonary granulomas develop. Characteristics such as increased fibrous encapsulation and development of a necrotic core appear to be similar to granulomas in cattle. However, mineralisation may not be a consistent feature, suggesting some species-specific differences that should be further investigated. This study also demonstrated that immunohistochemistry is a practical method for further characterisation of local immune responses to M. bovisinfection in buffalo. Further research with a larger sample set will be informative for understanding local and associating systemic immune responses to bTB in buffaloes.
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    The complete genome sequence of the African buffalo (Syncerus caffer)
    (BioMed Central, 2016-12-07) Glanzmann, Brigitte; Moller, Marlo; Le Roex, Nikki; Tromp, Gerard; Hoal, Eileen G.; Van Helden, Paul D.
    Background: The African buffalo (Syncerus caffer) is an important role player in the savannah ecosystem. It has become a species of relevance because of its role as a wildlife maintenance host for an array of infectious and zoonotic diseases some of which include corridor disease, foot-and-mouth disease and bovine tuberculosis. To date, no complete genome sequence for S. caffer had been available for study and the genomes of other species such as the domestic cow (Bos taurus) had been used as a proxy for any genetics analysis conducted on this species. Here, the high coverage genome sequence of the African buffalo (S. caffer) is presented. Results: A total of 19,765 genes were predicted and 19,296 genes could be successfully annotated to S. caffer while 469 genes remained unannotated. Moreover, in order to extend a detailed annotation of S. caffer, gene clusters were constructed using twelve additional mammalian genomes. The S. caffer genome contains 10,988 gene clusters, of which 62 are shared exclusively between B. taurus and S. caffer. Conclusions: This study provides a unique genomic perspective for the S. caffer, allowing for the identification of novel variants that may play a role in the natural history and physiological adaptations.
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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 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
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    SNP discovery and selection in Cape buffalo for bTB association study, using an African buffalo genome reference
    (Stellenbosch : Stellenbosch University, 2021-03) Engelbrecht, Marius Adriaan; Kinnear, Craig; Moller, Marlo; Glanzmann, Brigitte; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: The African buffalo (Syncerus caffer) is an important herd-based bovid in Africa, which is ubiquitous across almost the entire continent. These animals also act as a maintenance host for the ever-present threat that is bovine tuberculosis (bTB). The animal facilitates the spread and continued existence of the health problem that is bTB amongst wildlife and domestic cattle populations throughout Africa, causing problems in terms of conservation and economic loss. The disease is endemic to the southern part of Africa, especially South Africa, where two major national parks, The Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP), are host to it. There are also spill-over events of the disease from animals to humans, which is especially problematic in South Africa where tuberculosis (TB) in humans is already a major health concern. This study aimed to use 40 high-quality low-coverage African buffalo whole genome sequences in conjunction with a species-specific reference genome to create a panel of single nucleotide polymorphisms (SNPs) for use in further research in genetic association in buffalo bTB susceptibility. The sequences were from 40 Cape buffalo from 4 South African national parks, namely KNP, HiP and two bTB unexposed regions, the Mokala National Park (MNP) and Addo Elephant National Park (AENP). From this we produced a panel of 3698 high quality SNPs across 26 immune related genes in the African buffalo genome. One hundred and forty-three of these SNPs in three genes from the panel was used in a preliminary targeted association test with bTB exposure, which produced 10 SNPs associated with TB exposure. This may aid in future research and subsequent association studies.