MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.
Key words: Mycobacterium bovis -spoligotyping -MIRU-VNTR typing -bovine tuberculosis -molecular epidemiologyGenotyping of M. bovis from Brazil • Patrícia Martins Parreiras et al.
65While IS6110-RFLP lacks resolution in this species, spoligotyping has been demonstrated to be a fast and cost-effective method for first-line typing (Haddad et al. 2004). The MIRU-VNTR typing procedure has been extensively evaluated in M. tuberculosis and the recent data available about this method on M. bovis strains confirmed its considerable power of discrimination (Sola et al. 2003, Michel et al. 2008, 2010. Smith et al. (2006) inferred that a combination of spoligotyping and VNTR typing resulted in the simplest and most cost-effective method for routine molecular typing and epidemiological tracing of bovine TB in Great Britain.Some previous studies have successfully employed techniques such as IS6110-RFLP and spoligotyping to genotype M. bovis strains isolated from cattle in Brazil (Zanini et al. 2001, Rodriguez et al. 2004), but as far as we know, no data exist on genotyping of M. bovis by combining spoligotyping and MIRU-VNTR typing in our country. We aimed to use spoligotyping and MIRU-VNTR typing to assess the genetic diversity of Brazilian M. bovis isolates.
MATERIALS AND METHODS
Sampling and conventional procedures -During 2001and 2002, tissue samples from cattle with lesions suggestive of TB were collected immediately after slaughter at the time of inspection in abattoirs. The study samples were collected in abattoirs that followed slaughter inspection procedures from the states of Amazonas (AM), Paraíba (PB), Distrito Federal (DF), Mato Grosso do Sul (MS), São Paulo, Santa Catarina (SC) and Minas Gerais (MG). The sampled specimens from respective carcasses were pooled, put on ice and sent to the Laboratório Nacional Agropecuário de Minas Gerais (Pedro Leopoldo, MG), where they were processed for inoculation in Stonebrink culture medium (Mota 1985). The cultures were incubated at 37ºC and verified for bacterial growth every week for a period of at least two months. Bacterial isolates were submitted to standard procedures for differentiation of certain species of the MTBC, including the evaluation of niacin production, nitrate reductase activity, Tween hydrolysis, catalase activity at 68ºC and resis...
The main cause of pulmonary tuberculosis (TB) is infection with
Mycobacterium tuberculosis (MTB). We aimed to evaluate the
contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in
patients from the state of Rondônia using respiratory samples and
epidemiological data from TB cases. Mycobacterium isolates were identified using
a combination of conventional tests, polymerase chain reaction-based restriction
enzyme analysis of hsp65 gene and hsp65 gene
sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded
bacterial cultures, including 369 cases positive for MTB and 75 cases positive
for NTM. Within the latter group, 14 species were identified as
Mycobacterium abscessus, Mycobacterium
avium, Mycobacterium fortuitum,
Mycobacterium intracellulare, Mycobacterium
gilvum, Mycobacterium gordonae,
Mycobacterium asiaticum, Mycobacterium
tusciae, Mycobacterium porcinum,
Mycobacterium novocastrense, Mycobacterium
simiae, Mycobacterium szulgai,
Mycobacterium phlei and Mycobacterium
holsaticum and 13 isolates could not be identified at the species
level. The majority of NTM cases were observed in Porto Velho and the relative
frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately
half of the TB subjects with NTM, a second sample containing NTM was obtained,
confirming this as the disease-causing agent. The most frequently observed NTM
species were M. abscessus and M. avium and
because the former species is resistant to many antibiotics and displays
unsatisfactory cure rates, the implementation of rapid identification of
mycobacterium species is of considerable importance.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.