Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

Romero, Beatriz; Aranaz, Alicia; Sandoval, Ángel; Álvarez, Julio; Juan, Lucía de; Bezos, Javier; Sánchez, Celia; Galka, Margarita; Fernández, Pilar; Mateos, Ana; Domı́nguez, Lucas

doi:10.1016/j.vetmic.2008.04.032

Cited by 69 publications

(67 citation statements)

References 34 publications

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“…The six VNTR loci were selected based on an initial evaluation of a panel of 24 loci in 60 M. bovis isolates (unpublished data). The allelic diversities recorded for loci QUB 11b, 2165, MIRU 26, and 4052 were very similar to findings in other studies in Northern Ireland (17), Italy (2), Spain (15), and the United States (13). The allelic (17).…”

Section: Discussionsupporting

confidence: 88%

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

McLernon¹,

Costello²,

Flynn³

et al. 2010

J Clin Microbiol

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Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.The development of molecular techniques for differentiation of Mycobacterium bovis isolates has been of considerable benefit in epidemiological studies. Typing methods that have been commonly used include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (7,19).RFLP analysis of M. bovis isolates has commonly utilized polymorphism of the insertion sequence IS6110 and repetitive DNA elements such as the polymorphic GC-rich sequence (PGRS) and the direct-repeat (DR) region. Analysis of polymorphism of IS6110, the PGRS, and the DR region in combination has provided a high level of discrimination between strains (7, 19). REA has been widely used in New Zealand and has also given excellent resolution of strains (4). However, both RFLP analysis and REA require relatively large quantities of DNA and are laborious and time-consuming procedures. Complex banding patterns make analysis and interlaboratory comparisons difficult. Spoligotyping is a PCR-based typing method that reveals the presence or absence of unique spacer sequences located between the direct-repeat sequences of the DR region (12). It is a relatively easy procedure to perform, and the results can be expressed in a digital format. However, spoligotyping does not differentiate M. bovis strains t...

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Section: Discussionsupporting

confidence: 88%

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

McLernon¹,

Costello²,

Flynn³

et al. 2010

J Clin Microbiol

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“…Although we could not exclude or confirm any of these possibilities, we think that these are likely due to mixed infections, because the presence of double bands affected two or three markers, except for in one isolate. This effect has been previously described in M. bovis isolates from high-prevalence settings (9).…”

Section: Fig 1 Minimum Spanning Trees (Msts) Of the 115 Mycobacteriumsupporting

confidence: 70%

“…The occurrence of double bands in the MIRU-VNTR analysis, as observed in three isolates in this study, might be due to mixed infection (9,39) or to microevolutionary events (40). Although we could not exclude or confirm any of these possibilities, we think that these are likely due to mixed infections, because the presence of double bands affected two or three markers, except for in one isolate.…”

Section: Fig 1 Minimum Spanning Trees (Msts) Of the 115 Mycobacteriummentioning

confidence: 63%

“…For the first panel, VNTR typing was carried out targeting nine VNTR loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232) (5,8,(20)(21)(22). Six of these markers (ETR-A, ETR-B, ETR-D, QUB11a, QUB11b, and QUB3232) were recommended for typing M. bovis by the Veterinary Network of Laboratories Researching into Improved Diagnosis and Epidemiology of Mycobacterial Diseases (VENoMYC) Consortium (EU Coordination Action SSPE-CT-2004-501903 [23]), and the other three loci were previously used for typing Spanish strains (ETR-E [24,25], MIRU26 [24], and QUB26 [9]). PCR was performed using the HotStarTaq DNA polymerase kit (Qiagen) in a Bio-Rad MyCycler thermal cycler.…”

Section: Genotyping Methods (I) Spoligotypingmentioning

confidence: 99%

“…However, in some geographical settings, this cost-effective high-throughput technique does not offer adequate discrimination (2,3,4) and is thus complemented with mycobacterial interspersed repetitive-unit-variablenumber tandem-repeat (MIRU-VNTR) typing (5,6). The MIRU-VNTR technique has been evaluated in many countries using different sets of makers (2,4,(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

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confidence: 99%

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Splitting of a Prevalent Mycobacterium bovis Spoligotype by Variable-Number Tandem-Repeat Typing Reveals High Heterogeneity in an Evolving Clonal Group

et al. 2013

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Bovine tuberculosis (Mycobacterium bovis) remains a concern in Spain; although herd prevalence has been considerably reduced since the systematic implementation of the national eradication program in the 1990s, infection still affected 1.33% of herds in 2011 (per the Ministry of the Environment and Rural and Marine Affairs). In consideration of the economic and public health implications of this zoonosis, tracing the source of infection by genotyping has become a key element within the program. The most widely used technique to type M. bovis isolates is direct variable repeat (DVR) spoligotyping (1). However, in some geographical settings, this cost-effective high-throughput technique does not offer adequate discrimination (2, 3, 4) and is thus complemented with mycobacterial interspersed repetitive-unit-variablenumber tandem-repeat (MIRU-VNTR) typing (5, 6). The MIRU-VNTR technique has been evaluated in many countries using different sets of makers (2, 4, 7-15).In recent surveys in the Iberian Peninsula, SB0121 was found to be the most frequent spoligotype of M. bovis (26.3% in Portugal [16] and 27.9% in Spain [17]). These studies showed that high levels of discrimination could be achieved by spoligotyping (discriminatory index [D] ϭ 0.89 and 0.87 in Portugal and Spain, respectively), although subsequent studies from Portugal found that discrimination actually increased with MIRU-VNTR typing (12,13).In this study, we used nine MIRU-VNTR markers to type a panel of randomly selected Spanish M. bovis isolates with the spoligotype SB0121. The allelic diversity and overall discriminatory power of the technique were evaluated, using several different formats. Additionally, MIRU-VNTR was applied to all the infected animals from farms infected with M. bovis SB0121 to evaluate if heterogeneity was underdetected. MATERIALS AND METHODSMycobacterial strains. The study was carried out in M. bovis isolates with spoligotype SB0121 that were sampled from across Spain between 1997 and 2010. To evaluate the discriminatory power of MIRU-VNTR, we used a panel (n ϭ 115) (see Table S1 in the supplemental material) comprising 111 isolates selected randomly from 3,863 M. bovis SB0121 isolates from the Spanish strain collection at the VISAVET Health Surveillance Centre affecting animals plus four additionally selected M. bovis isolates from less frequently affected species. The isolates originated from cattle (Bos taurus) (n ϭ 98), goats (Capra aegagrus hircus) (n ϭ 2), wild boars (Sus scrofa) (n ϭ 7), red deer (Cervus elaphus) (n ϭ 2), fallow deer (Dama dama) (n ϭ 2), badgers (Meles meles) (n ϭ 2 [18]), and domestic pigs (Sus scrofa domestica) (n ϭ 2).To evaluate potential heterogeneity within the farms infected by M. bovis SB0121, we randomly selected 15 farms from 1,342 cattle farms from which isolates were included in the strain collection to design a second panel that comprised all the SB0121 isolates obtained from infected animals from these farms (total n ϭ 176; number of isolates per farm, 2 to 31).

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Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil

Carneiro

Pasquatti

Takatani³

et al. 2019

Veterinary Medicine & Sci

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The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.

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Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

Cited by 69 publications

References 34 publications

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

Splitting of a Prevalent Mycobacterium bovis Spoligotype by Variable-Number Tandem-Repeat Typing Reveals High Heterogeneity in an Evolving Clonal Group

Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil

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