Material and Methods
Study Area and Sample Collection
Field work was carried out in 14 sites (Figure 2) in two Pantanal sub-regions (Leque-do-Taquari
and Nhecolândia) (Da Silva and Abdon, 1998, Hamilton, et al., 1996). The capture sites were
registered by GPS (Global Position System). Twenty six animals were captured with attempts to
capture them in the places shared by cattle; the locations of the sites where only the AFBpositive samples were collected are shown in Table 1.
Pampas deer and feral pigs were captured using anesthetic darts according to the Animal Care
and Use Committee (Committee, 1998), following the guidelines and provisions of IBAMA,
Brazil (license number 20029-1; 35296-1). After being completely sedated, a bacteriologicalgrade sterile swab was introduced into the oropharyngeal region through the open mouth of
the pampas deer; a sample was collected and stored for later analysis. The retropharyngeal
lymph nodes from feral pigs were removed from euthanized animals through necropsy. Coatis
were captured using Tomahawk traps, after which they were anesthetized and oropharyngeal
swab samples were collected.
Samples and DNA extraction
Retropharyngeal lymph nodes from feral pigs were homogenized in 2mL of sterile water,
centrifuged at 3000 rpm for 15 min, the supernatant discharged, and the pellet resuspended
with 2mL of sterile water. Briefly, samples were decontaminated by Petroff’s method, and
cultured in Stonebrink medium. Oropharyngeal swabs from pampas deer and coati were
directly cultured on Stonebrink medium. All samples were incubated at 37°C for at least 90
days. Cells of each culture were checked for acid fastness by Ziehl-Neelsen staining. From 26
samples, only 14 were acid-fast bacilli (AFB). For DNA extraction, an AFB positive colony was
suspended in 1.5 mL TE buffer. Cells were inactivated and lysed by heat in a dry bath at 90°C
for 30 minutes, and then frozen at −20°C.
PCR amplification and sequencing
A 441-bp fragment of the hsp65 gene was amplified using previous published primers
(SHINNICK, 1987, TELENTI, et al., 1993). A 10 μl sample of the amplified product was subjected
to electrophoresis and the DNA band was excised from the gel, and purified using AxyPrep
DNA Gel Extraction Kit (Axygen Biosciences, USA). The PCR product was used as a template for
sequencing using the PCR primers with the BigDye Terminator Sequencing Kit (Applied
Biosystems, USA), and the reaction products were analyzed on an ABI 3130 (Applied
Biosystems, USA). The sequences of both strands were determined. Multiple sequence
alignment was carried out with Clustal W using MEGA 5.1 software (Tamura, et al., 2011). The
putative identities of the aligned sequences were determined using Nucleotide Basic Local
Alignment Search Tool (BLASTn) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The identity of each
sample was considered valid when higher than 98%. To construct a phylogenetic tree several
reference strains were obtained from GenBank: M. tuberculosis (GenBank accession number:
AY299144.1), M. avium and M. bovis (reference strain from Laboratório de Biologia Molecular
do LANAGRO/MG). The corresponding sequence of Nocardia farcinica ATCC 3308 (GenBank
accession number: DQ789016.1) was added as the phylogenetic outgroup. Based on these
amino acid sequences, a phylogenetic tree was constructed by the neighbor-joining method
using MEGA software. The degree of confidence in phylogenetic branching was assessed by
using 1,000 bootstrap resamplings.
MIRU-VNTR
One sample that could not be accurately identified using the hsp65 gene fragment
amplification method was amplified with 24 oligonucleotides related to MIRU-VNTR, as
described previously (Supply, 2005, Supply, et al., 2006). The fragment length of amplified
product was compared to the molecular marker, and the base pair number for each locus was
determined. The genetic profile of each isolate was compared with the reference database of
the web application MIRU-VNTRplus (http://www.miru-vntrplus.org). Reference strains from
the web application were used to construct a phylogenetic tree by the neighbor-joining
method. The strain names and reference numbers used were: M. bovis (7072/01, 751/01,
4258/00, 7540/01, 951/01, 8490/00); M. tuberculosis Beijing (3272/02, 3309/02, 4436/02); M.
tuberculosis Cameroon (5400/02), M. tuberculosis H37Rv (9679/00); M. africanum 1 (1473/02,
8303/02, 5398/02); M. africanum 2 (5383/02, 70485/01, 9550/00); M. caprae (8986/99,
11443/99, 9577/99); M. canettii (3040/99); M. microti (8473/00, 1479/00) and M. pinnipedi (
7739/01).