Isolation and genetic analysis of mycobacteria from suspect tuberculous lesions in slaughtered cattle from Wolaita, Ethiopia

Abstract

Bovine tuberculosis (bTB), caused by Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTBC), is a significant concern for livestock and public health in Ethiopia. This study aimed to isolate and genetically characterize the causative agents of bTB in cattle from four abattoirs in the Wolaita zone of Ethiopia. A total of 2,251 cattle were examined post-mortem, and suspect tuberculous lesions were identified in 122 animals. From these animals, 180 tissue samples were collected and processed for bacteriological culture and genetic analysis, including the Loopamp^™ commercial loop-mediated isothermal amplification kit, PCR targeting RD4 and RD9 loci and whole-genome sequencing (WGS). Bacteriological culture using mycobacteria growth indicator tube and Lowenstein-Jensen media ultimately identified 18 culture-positive samples, with WGS confirming M. bovis in lesions from four animals and M. tuberculosis in lesions from one animal. The M. bovis and M. tuberculosis isolates showed genetic similarity to previously identified MTBC lineages in Ethiopia. The presence of M. tuberculosis in cattle raises concerns about human-to-animal transmission. Additionally, non-tuberculous mycobacteria were isolated from lesions from multiple animals. Our study genetically characterized bacteria from suspect tuberculous lesions and provides the research community with new genome data for Ethiopian isolates of M. bovis and M. tuberculosis.

Keywords: Mycobacterium bovis; bovine tuberculosis; culture; lesion; post-mortem inspection; region of difference (RD) typing; whole-genome sequencing.

Fig. 1.. RD9 PCR results. Samples were tested by PCR for the presence or absence of the RD9 locus. Samples with RD9 present (i.e. M. tuberculosis) gave a 396 bp product, while samples that had RD9 deleted (i.e. M. bovis) gave a 575 bp product. (a) Lanes: 1, 100 bp molecular weight marker; 2, SH19; 3, SH20; 4, SH21; 5, SH23; 6, SH24; 7, SH25; 8, SH26; 9, SH05; 10, SH10; 11, SH14. (b) Lanes: 1, 100 bp molecular weight marker; 2, M. tuberculosis H37Rv control; 3, distilled water (no template) control; 4, M. bovis BCG control; 5, SH13; 6, A004_181; 7, SH15; 8, SH16; 9, SH17; 10, SH18.

Fig. 2.. RD4 PCR results. Samples were tested with PCR targeting the RD4 locus. If RD4 was present (i.e. M. tuberculosis), a product of 334 bp would be produced, while a product of 566 bp indicated RD4 was deleted (i.e. M. bovis). Lanes: 1, 100 bp molecular weight ladder; 2, SH05; 3, SH10; 4, SH14; 5, SH13; 6, SH15; 7, SH16; 8, SH17; 9, SH18; 10, 100 bp ladder; 11, SH19; 12, SH20; 13, SH21; 14, SH23; 15, SH24; 16, SH25; 17, SH26; 18, M. tuberculosis H37Rv control; 19, M. bovis BCG control; 20, distilled water (no template) control.

Fig. 3.. Phylogeny of M. bovis strains in this study and representative samples from Almaw et al. [29]. Tips are coloured by region of isolation, while to the right are heat maps showing the sub-lineage and spoligotype (SB types, Mbovis.org), respectively. Datasets used for this analysis are available via Zenodo (https://doi.org/10.5281/zenodo.13379235).

Fig. 4.. Phylogeny of M. tuberculosis strains SH13 and SH24 shown with representative M. tuberculosis lineages and MTBC species. Tips are coloured by species with the heat map on the right coloured by country of isolation. The dataset with sample accessions used for this analysis is available via Zenodo (https://doi.org/10.5281/zenodo.13379235).