Mhlophe Sinegugu Kholeka, Clarke Charlene, Ghielmetti Giovanni, Matthews Megan, Kerr Tanya Jane, Miller Michele Ann, Goosen Wynand Johan
Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, SAMRC Centre for Tuberculosis Research, Stellenbosch University, Stellenbosch, South Africa.
Section of Veterinary Bacteriology, Vetsuisse Faculty, Institute for Food Safety and Hygiene, University of Zurich, Zurich, Switzerland.
Front Vet Sci. 2025 Feb 7;12:1523628. doi: 10.3389/fvets.2025.1523628. eCollection 2025.
African buffaloes () are wildlife maintenance hosts of (), 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 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 ( = 19), originating from three herds with no recorded history of infection, to elucidate the possible cause of observed discordant immunological aTB test results. The DNA was extracted directly from the oronasal swabs, amplified using genus-specific PCRs, then amplicons were pooled and sequenced using Oxford Nanopore Technologies (ONT) long-read platform. 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.
非洲水牛（）是南非多个生态系统中动物结核病（aTB）病原体——牛分枝杆菌（）的野生动物维持宿主。除了作为关键物种的作用外，这些动物作为高价值物种对南非经济至关重要。在南非，控制aTB依赖于将分枝杆菌培养作为确诊的金标准，单剂量皮内比较颈侧试验（SICCT）和Bovigam™检测作为经过验证的细胞介导免疫检测方法用于检测。然而，这些方法并非没有缺点，在某些非结核分枝杆菌（NTM）干扰下，辨别真阳性结果的能力欠佳。本研究采用一种不依赖培养的方法，使用从19头非洲水牛采集的口鼻拭子，这些水牛来自三个没有aTB感染记录史的牛群，以阐明观察到的免疫aTB检测结果不一致的可能原因。直接从口鼻拭子中提取DNA，使用分枝杆菌属特异性PCR进行扩增，然后将扩增子合并并使用牛津纳米孔技术（ONT）长读长平台进行测序。在19个样本中的8个样本中鉴定出了牛分枝杆菌复合群DNA以及各种NTM物种。所描述的方法有助于对水牛口鼻部分枝杆菌群落进行更有力的研究。这些发现凸显了在复杂样本中准确区分分枝杆菌物种的价值，尤其是在高价值动物中，以促进免疫检测结果的准确解读和aTB的管理。
