3.2 Phylogenetic reconstruction of M. bovis from the safari park
Out of the 19 M. bovis genomes obtained from deer, two were excluded because they presented only 35% and 43% coverage againstM. tuberculosis H37Rv. All remaining genomes were confirmed to beM. bovis based on the absence of RD4 (i.e. region is deleted) and presence of RD1 (i.e. region is not deleted). Interestingly, one genome (TB002S2L) presented 35.77% of heterogeneous SNPs, suggesting the existence of a mixed strain infection in a deer. The remaining 16M. bovis isolates from deer and both isolates obtained from both llamas were further used to construct a ML phylogenetic tree and a PCA using a core-SNP matrix (Figure 3).
The phylogenetic reconstruction suggests the existence of four distinct clusters circulating in the safari park. The most basal of these clusters is formed by a single M. bovis isolate from a deer (TB013S13L, purple cluster, Figure 3), while the three other clusters are formed by six M. bovis isolates from deer (blue cluster, Figure 3), 4 M. bovis isolates from llamas and deer (black cluster, Figure 3), and seven M. bovis from deer (green cluster, Figure 3). The PCA analysis indicates the separation of at least three clusters, while two of them appeared very closely related (green and black), as also depicted in the phylogenetic tree (Figure 3, Appendix Figure 1). All M. bovis isolates of the safari park were identified as carrying the European 2 marker, being thus identified as lineage Lb3 according to recent classification (14 ). Interestingly, the spoligotype pattern was the same, SB1401, for allM. bovis genomes from deer, while both llama isolates had spoligotype pattern SB0295 (Figure 3).