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 (<i>Syncerus caffer</i>) are wildlife maintenance hosts of <i>Mycobacterium bovis</i> (<i>M. bovis</i>), 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 <i>M. bovis</i> 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 (<i>n</i> = 19), originating from three herds with no recorded history of <i>M. bovis</i> infection, to elucidate the possible cause of observed discordant immunological aTB test results. The DNA was extracted directly from the oronasal swabs, amplified using <i>Mycobacterium</i> genus-specific PCRs, then amplicons were pooled and sequenced using Oxford Nanopore Technologies (ONT) long-read platform. <i>Mycobacterium tuberculosis</i> 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.