Evidence of Mycobacterium bovis DNA in Shared Water Sources at Livestock-Wildlife-Human Interfaces in KwaZulu-Natal, South Africa

Megan C Matthews ¹ Debbie Cooke ¹ Tanya J Kerr ¹ Andre G Loxton ¹ Robin M Warren ¹ Giovanni Ghielmetti ^1,2 Elizabeth M Streicher ¹ Michele A Miller ¹ Wynand J Goosen ^1*

• ¹ Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa
• ² Sections of Veterinary Bacteriology and Avian diseases, Institute for Food Safety and Hygiene, University of Zurich, Zurich, Switzerland

The Mycobacterium tuberculosis complex (MTBC) includes M. bovis, which primarily affects animal hosts, but can also cause zoonotic infections in humans. Direct contact with infected animals or animal products is the major transmission pathway. However, recent research suggests that M. bovis can be shed into the environment, potentially playing an under-recognized role in the spread of the pathogen. Further investigation into indirect M. bovis transmission, employing a One Health approach, is needed to evaluate its epidemiological significance. However, current methods are not optimized for identifying M. bovis in complex environmental samples. Nevertheless, in a recent study, a combination of molecular techniques, including next generation sequencing (NGS), was able to detect M. bovis DNA in the environment to investigate epidemiological questions. The aim of this study was therefore to apply a combination of culture-independent methods, including targeted NGS (tNGS), to detect pathogenic mycobacteria, including M. bovis, in water sources in a rural area of KwaZulu-Natal (KZN), South Africa. This area was selected based on the high burden of MTBC in human and animal populations. Water samples from 63 sites were screened for MTBC DNA by extracting DNA and performing hsp65 PCR amplification, followed by Sanger amplicon sequencing (SAS). Sequences were compared to the National Centre for Biotechnology Information (NCBI) database for genus or species level identification. Samples confirmed to contain mycobacterial DNA were subjected to multiple PCRs (hsp65, rpoB, MAChsp65), and sequencing with Oxford Nanopore Technologies (ONT) tNGS. The ONT tNGS consensus sequences were compared to an in-house curated database to identify mycobacteria to genus, species, or species complex (e.g. MTBC) level for each sample site. Additional screening for MTBC DNA was performed using the GeneXpert ® MTB/RIF Ultra (GXU) qPCR assay. Based on GXU, hsp65 SAS and ONT tNGS results, MTBC DNA was present in 12 out of the 63 sites. The presence of M. bovis DNA was confirmed at 4 of the 12 sites using downstream PCR-based methods. However, additional studies are required to determine if environmental M. bovis is viable. These results support further investigation into the role that shared water sources may play in TB epidemiology.