MATERIAL AND METHODS
The study included 28 bovines and two buffaloes that had been attended
at the Bovine Clinic of Garanhuns/ UFRPE, presenting clinical symptoms
suggestive for tuberculosis. The animals were submitted to clinical
examination, being the information, including epidemiological, that was
annotated in clinical records. Among the information present in the
anamnesis provided by the owners, common to most animals, were
progressive weight loss, dry cough, and decreased milk production.
According to the evolution/severity of the clinical cases and the result
of the allergic-skin test, the animals were euthanized according to the
current legislation (Brazil, Ministry of Agriculture, Livestock and
Supply. Normative Instruction n. 19, 10 of October, 2016) and submitted
to anatomopathological examination.
Fragments of organs with lesions characteristic of granulomas were
collected for histopathological examination and lymph nodes with lesions
for microbiological culture.
The samples for bacteriology were stored in a freezer (-80°C) for
further processing while for histopathological evaluation, fragments
were fixed in 10% buffered formaldehyde, processed, and stained with
hematoxylin and eosin (HE). Granulomas from all 30 bovines were
collected and sent for microbiological culture an sample processing and
culture conditions favoring isolation of M. tuberculosis var.
bovis were carried out following
the recommendations of Franco et
al. (2017). Samples were minced and decontaminated according to the
Petroff method, inoculated on Lowenstein-Jensen and Stonebrink medium
and incubated at 37°C for 90 days.
Nucleic acid obtained of the cells was performed by thermolysis.
Molecular identification to the Mycobacterium species was
performed by PCR amplification of a 1020 bp fragment of the gyr B
gene as described by Chimara et al. (2004), Franco et al. (2017). In the
reaction, 1 µL of DNA (20 ng), 47 µL of Master Mix (1x) were used
(Thermo Scientific, Waltham, MA, USA) and 10 pMol of each primers MTUBf
(5’ TCGGACGCGTATGCGATATC 3’) and MTUBr (5’ACATACAGTTCGGACTTGCG 3’)
[DNA Express Biotecnologia LTDA, Brazil]. The cycling profile
consisted of denaturation at 95°C for 10 minutes, followed by 35
amplification cycles at 94°C for 1 minute, 65°C for 1 minute and 72°C
for 1.5 minutes, and final extension at 72°C for 10 minutes. The
amplification and fragment size was confirmed by electrophoresis in
agarose gel (1%) stained with GelRed ™ (Biotium, Hayward, CA, USA)
using a 100 bp molecular marker (DNA Express Biotecnologia LTDA). Then,
10 μL of the amplified product was submitted to Restriction Fragments
Length Polymorphism (RFLP) through digestion by restriction enzymesRsa I, Taq I and Sac II (Thermo Scientific, Waltham,
MA, USA), following the manufacturer’s recommendations. The generated
fragments were separated on 2% agarose gel stained with GelRed™ using
50 bp and 100 bp molecular markers (DNA Express Biotecnologia LTDA).
After electrophoresis, the gels were photographed in photo-documentation
equipment (2UV Transilluminator UVP) and restriction patterns compared
to those described by Chimara et al. (2004).
Spoligotyping was performed as described by Kamerbeek et al.
(1997). For amplification of the DR region, 20 μM of each primer DRa 5´
GGTTTTGGGTCTGACGAC 3´ (5’ biotinylated) and DRb (5´ CCGAGAGGGGACGGAAAC
3´), MyTaq Mix (12.5 μL, 1 μl (20 ng) genomic DNA and ultra-pure water
(9.5 μL) were submitted to PCR in a final volume of 25 µL.
The MIRU-VNTR typing using a combination of 24 loci was performed
according to Supply et al. (2006). In each PCR reaction 10 µL MyTaq Mix
(BIOLINE® ), 0.4 µL of each primer (20 mM), 2
µL of DNA (20 ng) and 7.2 µL of ultra-pure water were used in the final
volume 20 µL. Mycobacterium tuberculosis H37Rv DNA and water were
used as positive and negative controls, respectively.
The genetic profile based on spoligotyping of each isolate was
compared to those present in the international databasehttp://www.mbovis.org/andhttp://www.pasteur-guadeloupe.fr:8081/SITVITONLINE.
The 24-MIRU-VNTR patterns were compared to those present in the
MIRU-VNTRplus database deposited in the applicationhttp://www.miru-vntrplus.org/MIRU/index. The
Hunter-Gaston discriminatory index (HGDI) was performed to evaluate the
variability of the genotypes obtained by spoligotyping, and each of the
alleles of 24-MIRU-VNTR typing.