Amplification of a 500-basepair fragment from cultured isolates of Mycobacterium bovis

Corporación CorpoGen, Hospital San Juan de Dios, Santafé de Bogotá, D.C. Colombia.
Journal of Clinical Microbiology (Impact Factor: 3.99). 08/1999; 37(7):2330-2.
Source: PubMed


The presence of a 500-bp fragment which amplifies a region from the genome of Mycobacterium bovis (J. G. Rodriguez, G. A. Meija, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131-2138, 1995) was evaluated by carrying out PCR on 121 M. bovis isolates. The M. bovis strains, previously characterized by culture and biochemical tests, were isolated from cattle in different regions of Argentina, Mexico, and Colombia. Four additional strains isolated from sea lions that belong to the M. tuberculosis complex were also included in the study. All of the isolates tested were PCR positive, rendering the expected 500-bp band and giving a correlation of 100% with previous microbiological characterization. Southern blot analysis revealed a common band of 1, 800 bp and a polymorphic high-molecular-mass hybridization pattern. The results show that this assay may be useful for diagnosis and identification of M. bovis in cattle.

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    • "e genotipagem de Mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose Cabe destacar que os oligonucleotídeos JB21/JB22 foram inicialmente propostos como específicos para M. bovis (Rodríguez et al. 1995). No entanto, em estudo posterior, Rodríguez et al. (1999) verificaram que algumas cepas de M. tuberculosis também podiam ser amplificadas por eles. Há relatos de M. tuberculosis infectando bovinos (Ameni et al. 2011, Mbugi et al. 2012, Romero et al. 2011, Thakur et al. 2012). "
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    ABSTRACT: 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.
    Full-text · Article · Mar 2015 · Pesquisa Veterinária Brasileira
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    • "After growth in OK+pyruvate, colonies were molecularly typed by PCR. Briefly, purified mycobacterial DNA from colonies extracted as previously described [51] was used as template for PCR amplification of the following multi-copy insertion gene IS1081 (∼135 bp), present in the M. tuberculosis complex organisms (Fig. 1B) [52] and RvD1Rv2031c (∼500 bp) a polymorphic region of 2900 bp in the M. bovis genome which was not homologous in the genomes of M. tuberculosis and M. avium (Fig. 1C) [53].” "
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    ABSTRACT: Rational discovery of novel immunodiagnostic and vaccine candidate antigens to control bovine tuberculosis (bTB) requires knowledge of disease immunopathogenesis. However, there remains a paucity of information on the Mycobacterium bovis-host immune interactions during the natural infection. Analysis of 247 naturally PPD+ M. bovis-infected cattle revealed that 92% (n = 228) of these animals were found to display no clinical signs, but presented severe as well as disseminated bTB-lesions at post-mortem examination. Moreover, dissemination of bTB-lesions positively correlated with both pathology severity score (Spearman r = 0.48; p<0.0001) and viable tissue bacterial loads (Spearman r = 0.58; p = 0.0001). Additionally, granuloma encapsulation negatively correlated with M. bovis growth as well as pathology severity, suggesting that encapsulation is an effective mechanism to control bacterial proliferation during natural infection. Moreover, multinucleated giant cell numbers were found to negatively correlate with bacterial counts (Spearman r = 0.25; p = 0.03) in lung granulomas. In contrast, neutrophil numbers in the granuloma were associated with increased M. bovis proliferation (Spearman r = 0.27; p = 0.021). Together, our findings suggest that encapsulation and multinucleated giant cells control M. bovis viability, whereas neutrophils may serve as a cellular biomarker of bacterial proliferation during natural infection. These data integrate host granuloma responses with mycobacterial dissemination and could provide useful immunopathological-based biomarkers of disease severity in natural infection with M. bovis, an important cattle pathogen.
    Full-text · Article · Jan 2013 · PLoS ONE
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    • "Kurabachew et al. (2004) developed a multiplex PCR assay targeting 23 s rDNA and the oxyR gene to distinguish between M. tuberculosis and M. bovis, where M. tuberculosis strains produce both the 23 s rDNA and the oxyR amplicons, whereas only the 23 s rDNA amplicons are produced by M. bovis. Several other genes and insertion sequences have been targeted in attempts to develop PCR assays to differentiate M. bovis from other members of MTBC (Rodriguez et al. 1995, 1999; Shah et al. 2002; Prabhakar et al. 2004; Bakshi et al. 2005; Young et al. 2005). Furthermore, a specific deletion in the genome of M. bovis known as region of difference 4 (RD4) has been exploited in PCR assays. "
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