Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa
African buffaloes (Syncerus caffer) are wildlife maintenance hosts of Mycobacterium bovis (M. bovis), the causative agent of animal tuberculosis (aTB) in multiple ecosystems across South Africa. In addition to their role as keystone species, these animals are vital to South Africa’s economy as a hig...
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Frontiers Media S.A.
2025-02-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2025.1523628/full |
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| author | Sinegugu Kholeka Mhlophe Charlene Clarke Giovanni Ghielmetti Giovanni Ghielmetti Megan Matthews Tanya Jane Kerr Michele Ann Miller Wynand Johan Goosen Wynand Johan Goosen |
| author_facet | Sinegugu Kholeka Mhlophe Charlene Clarke Giovanni Ghielmetti Giovanni Ghielmetti Megan Matthews Tanya Jane Kerr Michele Ann Miller Wynand Johan Goosen Wynand Johan Goosen |
| author_sort | Sinegugu Kholeka Mhlophe |
| collection | DOAJ |
| description | African buffaloes (Syncerus caffer) are wildlife maintenance hosts of Mycobacterium bovis (M. bovis), the causative agent of animal tuberculosis (aTB) in multiple ecosystems across South Africa. In addition to their role as keystone species, these animals are vital to South Africa’s economy as a highly valuable species. Controlling aTB in South Africa relies on mycobacterial culture as the gold standard for M. bovis confirmation, with the single intradermal comparative cervical test (SICCT) and Bovigam™ assays as validated cell-mediated immunological assays for detection. However, these methods are not without their shortfalls, with a suboptimal ability to discern true positive results amidst certain non-tuberculous mycobacteria (NTM) interference. This study employed a culture-independent approach using oronasal swabs collected from African buffaloes (n = 19), originating from three herds with no recorded history of M. bovis infection, to elucidate the possible cause of observed discordant immunological aTB test results. The DNA was extracted directly from the oronasal swabs, amplified using Mycobacterium genus-specific PCRs, then amplicons were pooled and sequenced using Oxford Nanopore Technologies (ONT) long-read platform. Mycobacterium tuberculosis complex DNA, along with various NTM species, were identified in 8/19 samples. The methods described support a more robust interrogation of the buffalo oronasal mycobacteriome. These findings highlight the value of accurately distinguishing between mycobacterial species in complex samples, especially in high-value animals, to facilitate accurate interpretation of immunological test results and management of aTB. |
| format | Article |
| id | doaj-art-abb8bb718c3f4ea083400dadbaddb696 |
| institution | Kabale University |
| issn | 2297-1769 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Veterinary Science |
| spelling | doaj-art-abb8bb718c3f4ea083400dadbaddb6962025-02-07T06:49:42ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692025-02-011210.3389/fvets.2025.15236281523628Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South AfricaSinegugu Kholeka Mhlophe0Charlene Clarke1Giovanni Ghielmetti2Giovanni Ghielmetti3Megan Matthews4Tanya Jane Kerr5Michele Ann Miller6Wynand Johan Goosen7Wynand Johan Goosen8Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaSection of Veterinary Bacteriology, Vetsuisse Faculty, Institute for Food Safety and Hygiene, University of Zurich, Zurich, SwitzerlandDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaDivision of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South AfricaDepartment of Microbiology and Biochemistry, Faculty of Natural and Agricultural Sciences, University of the Free State, Bloemfontein, South AfricaAfrican buffaloes (Syncerus caffer) are wildlife maintenance hosts of Mycobacterium bovis (M. bovis), the causative agent of animal tuberculosis (aTB) in multiple ecosystems across South Africa. In addition to their role as keystone species, these animals are vital to South Africa’s economy as a highly valuable species. Controlling aTB in South Africa relies on mycobacterial culture as the gold standard for M. bovis confirmation, with the single intradermal comparative cervical test (SICCT) and Bovigam™ assays as validated cell-mediated immunological assays for detection. However, these methods are not without their shortfalls, with a suboptimal ability to discern true positive results amidst certain non-tuberculous mycobacteria (NTM) interference. This study employed a culture-independent approach using oronasal swabs collected from African buffaloes (n = 19), originating from three herds with no recorded history of M. bovis infection, to elucidate the possible cause of observed discordant immunological aTB test results. The DNA was extracted directly from the oronasal swabs, amplified using Mycobacterium genus-specific PCRs, then amplicons were pooled and sequenced using Oxford Nanopore Technologies (ONT) long-read platform. Mycobacterium tuberculosis complex DNA, along with various NTM species, were identified in 8/19 samples. The methods described support a more robust interrogation of the buffalo oronasal mycobacteriome. These findings highlight the value of accurately distinguishing between mycobacterial species in complex samples, especially in high-value animals, to facilitate accurate interpretation of immunological test results and management of aTB.https://www.frontiersin.org/articles/10.3389/fvets.2025.1523628/fullAfrican buffaloesculture-independent detectionMycobacterium tuberculosis complexoronasal swabsOxford Nanopore Technologiestargeted next generation sequencing |
| spellingShingle | Sinegugu Kholeka Mhlophe Charlene Clarke Giovanni Ghielmetti Giovanni Ghielmetti Megan Matthews Tanya Jane Kerr Michele Ann Miller Wynand Johan Goosen Wynand Johan Goosen Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa Frontiers in Veterinary Science African buffaloes culture-independent detection Mycobacterium tuberculosis complex oronasal swabs Oxford Nanopore Technologies targeted next generation sequencing |
| title | Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa |
| title_full | Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa |
| title_fullStr | Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa |
| title_full_unstemmed | Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa |
| title_short | Culture-independent detection of Mycobacterium tuberculosis complex DNA using targeted next generation sequencing in African buffalo (Syncerus caffer) oronasal swabs in South Africa |
| title_sort | culture independent detection of mycobacterium tuberculosis complex dna using targeted next generation sequencing in african buffalo syncerus caffer oronasal swabs in south africa |
| topic | African buffaloes culture-independent detection Mycobacterium tuberculosis complex oronasal swabs Oxford Nanopore Technologies targeted next generation sequencing |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2025.1523628/full |
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