Identification and genotyping of Mycobacterium bovis from positive cattle in skin test for tuberculosis in the State of Mato Grosso do Sul, Brazil

Spoligotyping was performed in the present study to genotype Mycobacterium bovis isolates obtained from tissues of cattle that were positive in the comparative intradermal tuberculin test (CITT) in the state of Mato Grosso do Sul (Brazil). Tissue samples from 13 positive cattle from different municipalities of the state were cultured using a Stonebrink medium. The resulting colonies were subjected to Ziehl-Neelsen staining and all isolates exhibited the staining characteristics of AFB. The 13 isolates of AFB were identified by means of a multiplex PCR (mPCR) assay. The hsp65 gene was targeted for the identification of Mycobacterium spp., whereas the IS6110 insertion sequence was targeted for the identification of the Mycobacterium tuberculosis complex (MTC) and the rvd1rv2031c region was explored for the detection of Mycobacterium bovis. The spoligotyping assay was performed to genotype mycobacterial isolates. Of the 13 cattle, seven had at least one lesion suggestive of tuberculosis in the retropharyngeal, parotid and lung lymph nodes or lung. The remaining six exhibited no lesions suggestive of the disease. In the mPCR, 11 of the 13 isolates (84.6%) were positive for Mycobacterium spp., 8/13 (61.5%) were positive for the MTC and 7/13 (53.8%) were positive for M. bovis. Based on the spoligotyping, eight isolates were grouped into three different groups of genotypes and one isolate exhibited an orphan type. Four isolates exhibited spoligotype pattern SB0121, while two isolates were associated with the pattern SB1145, another two were associated with pattern SB0881 and one was associated with pattern SB0140. Spoligotyping confirmed the genetic diversity present among isolates found in the state of Mato Grosso do Sul. In addition, SB0121 was confirmed as the predominant profile.