Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

The MIRU-VNTR is based on the size analysis of amplified fragments from multiple loci, determining the number of repetitions of each locus [29,30,31,32]. The analysis of the amplified fragment can be done manually by agarose gel electrophoresis [33] or automatically by capillary electrophoresis [34]. Each technique has its advantages and disadvantages that must be considered when choosing which to implement in the laboratory. Spoligotyping in combination with MIRU-VNTR analysis seems to be the best choice, since both have the advantage of being PCR-based, and, when combined, discriminatory power is improved [19].
In this context, a cross sectional study of molecular epidemiology was conducted for the characterization of M. bovis isolates circulating in the Brazilian Midwest and the comparison with M. bovis strains from other regions of Brazil and the world was performed.

Bacterial isolates and DNA extraction
The present study was based on a convenience sampling of BTB diagnosed between 2010 to 2013, at the National Agricultural Laboratory (LANAGRO/MAPA/BRASIL). A total of 37 M. bovis isolates were obtained from clinical samples taken from suspected BTB lesions from 37 animals that scored positive in the intradermal tuberculin test in the Brazilian Midwest region (Mato Grosso, Mato Grosso do Sul and Goiás). These isolates were previously identified by biochemical [26] and molecular tests [4]. DNA templates were extracted by the thermal lysis method [35] and purified using the commercial kit ChargeSwitch 1 PCR Clean-up kit (Invitrogen, CA, USA). DNA templates from M. bovis BCG and M. tuberculosis H37Rv were used as positive controls in the spoligotyping and MIRU-VNTR assays.

MIRU-VNTR typing
M. bovis strain typing was carried out by MIRU-VNTR automated in-house technique, according to De-Beer et al. (2012) [37] with modifications. The detection of 24-loci MIRU-VNTR labeled with fluorophores (6FAM™/green, VIC 1 /blue and NED™/yellow) was performed, as recommended by Supply et al. (2006) [32]. For each sample, eight PCRs were carried out, using three primer pairs (triplex-PCR) each for the simultaneous amplification of three distinct loci [32].
Tríplex-PCR was performed using 0.4 μl of each primer (Applied Biosystem, CA, USA), at the concentrations described by Supply et al. (2006)  PCR products (1 μl) were prepared for automated fragment reading on an optical plate-MicroAmp 1 Optical 96-well Reaction (Applied Biosystem, CA, USA) by adding 0.4 μl of the molecular marker GeneScan™ 1200 LIZ 1 Size Standard (Applied Biosystem), 8.6 μl Hidi formamide (Applied Biosystems) in a final volume of 10 μl. All mixtures were denatured at 95°C for 2 min and immediately cooled on ice. The fragment size of the amplicons was analyzed on a ABI 3130xl DNA sequence analyzer (Applied Biosystems) and the number of copies of each locus was determined by automated assignment using the GeneMapper 1 4.0 software (Applied Biosystems). In case of doubtful results, the length of the repeats was double checked by size fragment estimation as compared to a DNA ladder (50 and 100 bp). Aplicons from M. bovis BCG and H37Rv strains were compared with the reference table described by Supply et al. (2000) [31].

Allelic and genotypic diversity calculations
The Hunter-Gaston discriminatory index (HGDI) [38] was used to calculate the allelic diversity within each MIRU-VNTR locus and the genotypic diversities (discriminatory power) of the spoligotyping assays, 24-MIRU-VNTR and the combination of both methodologies.

Clustering analysis
The number and fragment length of the genotype clusters were introduced as numerical data into an Excel spreadsheet template and different criteria for definition of the clusters were used, such as the analysis of individual spoligotyping or combination of results from spoligotyping and MIRU-VNTR. Data were analyzed by the BioNumerics software 6.6 (Applied Maths, East Flanders, BE) in order to construct the similarity matrices and the dendrogram (unweighted pair-grouping method analysis algorithm-UPGMA).
Spoligotypes SB0121 and SB0295 differ by one spacer only in the DR (direct repeat) region (Table 1) and were presently responsible for 54% genotypes of the strains isolated from Midwestern Brazil. The small discrepancy in these spoligotypes may be associated with strains that have undergone genetic mutation, which may cause difficulties in BTB diagnostics through the conventional tuberculin test, adopted throughout the country for BTB control in cattle herds [19,52,53]. Infections caused by strains classified as SB0121 and SB0295 spoligotypes occurred in municipalities very near to each other and suggests a selection of these lineages in these geographic locations (Fig 1). Although spoligotype SB0140 was observed at a lower frequency (16.2%), it occurred in the three investigated states and was found with similar a frequency in São Paulo [54]. It has also been described throughout the four continents, in several countries, including Mexico [48,49,51], Argentina [49,55], Paraguay [55], Uruguay [55] Chile [49], France [41], Italy [43], Ireland [56,57], United Kingdom [58,59], South Africa [47] and Australia [56].
The SB0881 spoligotype was identified only in Mato Grosso do Sul (Fig 1) and is the third most prevalent in Brazil [49], having previously been reported in the country [20,39,40], and having also been shown to occur in Spain [45] and in France [41].
The SB1144 and SB1145 spoligotypes were identified in only two isolates each, the former in Goiás and the latter in Mato Grosso do Sul (Fig 1). These spolygotypes have only been found in Brazil. The spoligotype SB1145 is the most widely-distributed, being previously reported in São Paulo [54] [46] and in the United Kingdom [58,59]. SB1050 and SB1055 were reported in the Central and Latin Americas, particularly in Argentina, Paraguay, Uruguay, Mexico, Costa Rica (Mbovis.org) and Brazil [3,40,63].
The 24-loci MIRU-VNTR patterns and the combined genotyping results are displayed in Table 2 and in Fig 2. The UPGMA based similarity of the combined genotypes are also shown.
While the spoligotyping resulted in six clusters containing 89.2% (33/37) of the isolates, the 24 MIRU-VNTR typing also resulted in six cluster, albeit containing only 40.5% (15/37) of the M. bovis isolates and 22 orphan patterns, demonstrating higher discriminatory power of 24 MIRU-VNTR for typing of M. bovis strains circulating in the Midwest region (Tables 2 and 3 and Fig 2).
The allele diversity of each of the 24 MIRU-VNTR loci is presented in Table 4. Two loci (ETR-A and QUB 11b) were the most discriminatory (h = ! 0.50), while six presented moderate allelic diversity (MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21, QUB 26; h index between 0.33 to 0.49). Low allele diversity (h = 0.15) was observed for eight MIRUs and no diversity at all in another eight markers (Table 4). This means that eight MIRUs should be sufficient for the genotyping study of the M. bovis isolates from the Brazilian Midwest.
These results corroborate with earlier data, which showed high resolution of ETR-A, ETR-B and ETR-C in the genotyping of M. bovis isolates from the state of Rio de Janeiro [64]. High resolution of ETR-A and ETR-B was also observed in Chad  Previous studies conductes in the South and southeastern regions of Brazil analyzed the genetic variability of M. bovis isolates from 12 to 15-loci from MIRU-VNTR [19,40,64]. In the present study, the allelic diversity and, consequently, the discriminatory power of the 24 MIR-U-VNTR loci in a convenience sample obtained in the Midwest Braizlian region, from 2010 to 2013, were investigated for the first time.
Spoligotyping showed a discriminatory index of 0.810 (Table 3) 69]. The polymorphism of the strains is based on the variability of the number of copies of each repeating unit. The original MIRU-VNTR methodology included 12-loci was used in conjunction with spoligotyping for the first MTC genotyping. However, its discriminatory power was less than IS6110 RFLP [69,70]. Due to the low discriminatory power of the MIRU-VNTR 12-loci, current studies suggest the use of a set of 15-loci for molecular epidemiological studies and 24loci for phylogenetic studies [32]. Currently, the method has a high yield due to multiplex-PCR application using primers labeled with different fluorophores. This amplified material is subjected to capillary electrophoresis in an automatic sequencer, to estimate the size of the PCR product [71,72]. The advantage of automated typing by MIRU-VNTR is the fact that method is highly reproducible, faster and less laborious than the original methodology, yielding more reliable results because of the computerized analysis of the generated fluorescent signals. There is a consensus among different studies that, by associating the results of spoligotyping to those obtained by MIRU-VNTR, discrimination between strains is more effective, and, thus, the combination of methodology has been considered the best strategy for the molecular typing of M. bovis Herein, five clusters with 13 isolates (35%) (Fig 2) were observed and interestingly, strains with orphan patterns were found predominantly in the state of Goiás (10/24), besides the clustered strains. In the state of Mato Grosso, clusters "C", "D" and "E" were found, along with 5 orphan patterns. Finally, in the state of Mato Grosso do Sul, strains were clustered in "A" and "B" and nine of them showed orphan patterns. Epidemiologically related isolates are derived from the clonal expansion of a single precursor and as a result, have common characteristics that differ from those that are unrelated epidemiologically [64].
The great genetic heterogeneity of M. bovis observed in the Brazilian Midwest can be explained by the animal movement that occurs between different regions and farms, thus causing the spread of numerous M. bovis strains in the herds of the region. Another important point to consider is that the Midwest region of the country is a dry border with other Latin American countries, such as Bolivia and Paraguay, over a wide range of territory, thus allowing contact between herds of both countries, resulting in the transfer of M. bovis strains to Brazil, which can be retained in the Midwest region, or possibly migrate to other, more remote, regions.
In the present study, the association of spoligotyping and 24-MIRU-VNTR for the molecular characterization of M. bovis isolates from the Brazilian Midwest was carried out for the first time and indicated that BTB in this geographical region is caused by M. bovis isolates with high genetic diversity, which may hinder in vivo diagnosis, control and eradication of the disease. The characterization of M. bovis circulating genotypes in the geographical region aids in tracking and sanitizing remaining outbreaks of disease, since BTB has a low prevalence in this region of Brazil.

Conclusions
Ten spoligotypes are present in the Brazilian Midwest region. The combination of spoligotyping with the 24-MIRU analysis rendered five clusters and 24 orphan patterns, confirming the high genotypic diversity among M. bovis strains circulating in the Midwest Brazil. The presence of different M. bovis genotypes in this region suggests movement of animals between regions or different sources of infection. Thus, it is possible to conclude that BTB in the Brazilian Midwest is caused by multiple M. bovis strains.