Figure Legends
Figure 1. Location of the safari park (red drop) and the cities
of the cattle farms are represented by different colors on the map of
the Rio Grande do Sul state, Southern Brazil. The safari park is located
in the vicinity of Porto Alegre, the capital city of Rio Grande do Sul.
Pink: cattle herd from Não-Me-Toque; Midnight Green: Arroio do Tigre;
Zomp: Teutônia; Green: Charqueadas; Coral Pink: Estrela; Orange: Fazenda
Vila Nova; Cyan: Bom Retiro do Sul; Purple: Eldorado do Sul; Black:
Camaquã.
Figure 2. Timeline of the tuberculosis outbreak in the safari
park according to data collection of government authorities from the
state of Rio Grande do Sul, Brazil. Years in red represent zoo closure
(for visitors), following a court order in 2013. *Mycobacterium
bovis isolation performed in two llamas, and positive PCR in a third
animal. Isolation was not performed in the red deer. ** T+ S in deer, ND
+ N in a camel and an antelope. PPD: Purified protein derivative ofM. bovis – indicates that animals were subjected to tuberculin
test (comparative cervical test).
Figure 3. Phylogenetic reconstruction and principal component
analysis (PCA) of 18 Mycobacterium bovis genomes obtained from
the safari park. Mycobacterium bovis genomes starting with
“TB0” were obtained from deer (n= 16) and genomes starting with
“L” were obtained from llamas (n= 2). Phylogenetic clusters are
shown in the PCA using the same colors. NC_00295.4 is the reference
genome of M. bovis AF2122/97. Phylogenetic tree was constructed
from a core-SNP matrix using maximum likelihood in IQ-Tree with 1,000
bootstrap replicas. Strains of Mycobacterium caprae were used as
an outgroup. Lineages were identified according to Zimpel et al. (14).
Horizontal bar shows substitutions per nucleotide.
Figure 4. Bayesian phylogenetic tree of Mycobacterium
bovis genomes from the safari park (n=18) and cattle herds (n=71) of
Rio Grande do Sul. Phylogenetic tree was generated in BEAST v1.10.4,
using a core SNP-matrix. Horizontal bar indicates years before present.
Mycobacterium caprae strains are used as outgroup.
Figure 5. Minimum spanning tree of 18 Mycobacterium
bovis genomes from the safari park and 71 M. bovis from cattle herds of
Rio Grande do Sul state. Nodes are M. bovis genomes and edges
represent the number of SNPs between two genomes (number of SNPs
indicated on each edge). Nodes in orange represent possible transmission
links using a SNP cutoff of 12. Genomes of M. bovis starting with
TB0 (deer) and L (llama) are from the safari park, while genomes of M.
bovis starting with ERR are from cattle herds.
Appendix Table 1. List of Mycobacterium spp genomes and
respective accession numbers used in this study.
Appendix Table 2. Pairwise Single Nucleotide Polymorphism (SNP)
distance-matrix of Mycobacterium bovis genomes used in this
study.
Appendix Figure 1. Three-dimensional principal component
analysis (PCA) of Mycobacterium bovis genomes from the safari
park. Colors correspond to clusters depicted in Figure 1 of the article.
Component 1 = 66.58%, Component 2 = 24.36%, Component 3 = 3.88%. Red:Mycobacterium bovis AF2122/97 reference genome.
Appendix Figure 2. Single nucleotide polymorphism
(SNP)-distance between Mycobacterium bovis genomes of the safari
park (n=18) and of cattle (n=71) from the state of Rio Grande do Sul.
This matrix was generated from core-SNP matrix of all genomes using the
software PHYLOViZ 2.0 with default parameters. Appendix Table 2 shows
individual values. Mycobacterium bovis genomes starting with
“TB0” were obtained from cervids and genomes starting with “L” were
obtained from llamas. In green it is possible to see comparisons betweenM. bovis genomes of different lineages (Lb3 and Lb4) detected in
the dataset.
Appendix Figure 3 . Single nucleotide polymorphism
(SNP)-distance between Mycobacterium bovis genomes of the safari
park (n=18). This matrix was generated from core-SNP matrix of all
genomes using the software PHYLOViZ 2.0 with default parameters.Mycobacterium bovis genomes starting with “TB0” were obtained
from cervids and genomes starting with “L” were obtained from llamas.
Appendix Figure 4 . Comparison of the spoligotype patterns found
in cervids (SB1401) and llamas (SB0295) of the safari park. Spoligotype
patterns were detected using sequencing reads (fastq files) in SpoTyping
software.
Appendix Figure 5. Discordant spoligotype patterns of the
recent transmission links of Mycobacterium bovis detected using
genomics.