Molecular Discrimination ofMycobacterium bovisin São Paulo, Brazil

Abstract

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, is the most common agent of cattle tuberculosis, a zoonosis that causes losses in meat and milk production in several countries. In order to support epidemiological studies aimed at controlling the disease, several methods for molecular discrimination of M. bovis isolates have recently been developed. The most frequently used are spacer oligonucleotide typing (spoligotyping), mycobacterial interspersed repetitive units (MIRU), and exact tandem repeat (ETR), but they all have different discriminatory power. In the present study, allelic diversity was calculated for each MIRU and ETR locus, and the Hunter-Gaston discriminatory index (HGI) was calculated for spoligotyping, 10 MIRUs, and 3 ETRs, in 116 isolates of M. bovis obtained from cattle. The analysis of allelic diversity indicated that MIRUs 16, 26, and 27, and ETRs A, B, and C, showed the greatest diversity between the assayed loci. The HGIs for each of the techniques were: spoligotyping=0.738381; MIRU=0.829835; and ETR=0.825337. The associations of the methods' improved discriminatory power were: spoligotyping+MIRU=0.930585; spoligotyping+ETR=0.931034; and MIRU+ETR=0.953373. The greatest discriminatory power was obtained when the three techniques were associated (HGI=0.98051). Considering the analyses of the present study, spoligotyping should be the first method to be used because it differentiates M. bovis from the other members of the Mycobacterium tuberculosis complex. As the associations of MIRU and ETR with spoligotyping resulted in nearly identical HGIs, ETR seems to be the best choice after spoligotyping, because it is faster and more economical than MIRU. Finally, MIRU should be the last method used. In spite of this finding, the choice of the method used should be based on the discriminatory power necessary for the objective at hand.

Identificação e genotipagem de Mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul

“…Haddad et al (2004) (Rodriguez et al 2004, Rocha et al 2013). Nos estados de Minas Gerais, Mato Grosso do Sul e Paraíba sua presença também já foi relatada (Parreiras et al 2012).…”

“…O espoligotipo SB0140 também já foi detectado no estado de São Paulo (Rocha et al 2013), porém Zumárraga et al (2013) consideram sua frequência baixa no Brasil. É predominante nos países vizinhos, Argentina e Chile e já foi relatado no Reino Unido, Nova Zelândia, Austrália e Irlanda (Cousins et al 1998, Costello et al 1999, Zumárraga et al 1999, Haddad et al 2001, Smith et al 2011.…”

“…O espoligotipo SB0881, caracterizado pela ausência dos espaçadores 21 e 26-30 (Zumárraga et al 2013), foi relatado anteriormente em São Paulo (Rocha et al 2013) e é o terceiro mais encontrado no Brasil (Zumárraga et al 2013). Também já foi relatado na França e na Espanha (Haddad et al 2001, Rodríguez et al 2010.…”

Diagnosis of mycobacteria in bovine milk: an overview

“…The hyper variability of these repetitions in human and animal cells generate the Variable number tandem repeat (VNTR). In the diagnosis of mycobacterial isolates, the polymorphism may be analyzed by repetitions of sequences of nucleotides, or even variation in the number of repeating units 39 , 40 . The identification of 24 VNTR loci of M. tuberculosis genome received the name Mycobacterial Interspersed Repetitive Units (MIRU) 92 , and it has been used as a molecular marker for diagnosis of mycobacterial infections.…”

“…For NTM diagnosis, the hsp 65 gene is the most widely target used to classify this group of mycobacteria. When the target is M. bovis or M. tuberculosis, the MIRU-VNTR is a highly discriminatory method and an important tool in epidemiological research 39 .…”