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.