RESEARCH ARTICLE
Molecular Typing of Mycobacterium bovis
from Cattle Reared in Midwest Brazil
Ricardo César Tavares Carvalho
1,7
, Sidra Ezidio Gonçalves Vasconcellos
2
, Marina de
Azevedo Issa
4
, Paulo Martins Soares Filho
4
, Pedro Moacyr Pinto Coelho Mota
4
, Flábio
Ribeiro de Araújo
5
, Ana Carolina da Silva Carvalho
1,6
, Harrison Magdinier Gomes
2
,
Philip Noel Suffys
2,3
, Eduardo Eustáquio de Souza Figueiredo
7
*, Vânia Margaret
Flosi Paschoalin
1
1 Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro/RJ, Brasil,
2 Laboratório de Biologia Molecular Aplicado a Micobactérias, Instituto Oswaldo Cruz (IOC), Fundação
Oswaldo Cruz (FIOCRUZ), Rio de Janeiro/RJ, Brasil, 3 Mycobacteriology Unit, Tropical Institute of Medicine,
Antwerp, Belgium, 4 Laboratório Nacional Agropecuário (LANAGRO), Ministério da Agricultura, Pecuária e
Abastecimento (MAPA), Pedro Leopoldo/MG, Brasil, 5 Empresa Brasileira de Pesquisa Agropecuária
(EMBRAPA Gado de Corte), Campo Grande/MS, Brasil, 6 Universidade Federal do Rio de Janeiro (UFRJ)
—Campus Macaé, Macaé/RJ, Brasil, 7 Faculdade de Nutrição, Universidade Federal de Mato Grosso
(UFMT), Cuiabá/MT, Brasil
*
figueiredoeduardo@ufmt.br
Abstract
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 cir-
culating 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 iden-
tified 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 similari-
ties 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 move-
ment between regions, municipalities and farms, thus causing the spread of various M.
bovis strains in herds from Midwest Brazil.
PLOS ONE | DOI:10.1371/journal.pone.0162459 September 15, 2016 1 / 16
a11111
OPEN ACCESS
Citation: Carvalho RCT, Vasconcellos SEG, Issa
MdA, Soares Filho PM, Mota PMPC, Araújo FRd, et al.
(2016) Molecular Typing of Mycobacterium bovis from
Cattle Reared in Midwest Brazil. PLoS ONE 11(9):
e0162459. doi:10.1371/journal.pone.0162459
Editor: Srinand Sreevatsan, University of Minnesota,
UNITED STATES
Received: May 1, 2016
Accepted: August 23, 2016
Published: September 15, 2016
Copyright: © 2016 Carvalho et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors thank the Brazilian funding
agencies for support: PRONEM/FAPEMAT (472914/
2011) Dr. Eduardo Eustáquio de Souza Figueiredo;
CNPq/Universal 14/2014 (443235/2014-7) Dr. Flábio
Ribeiro de Araújo; EMBRAPA (02.13.16.002.00.00)
Dr. Flábio Ribeiro de Araújo; FUNDECT (085/2015)
Dr. Flábio Ribeiro de Araújo; CAPES/AUX-PE/
PROCAD (2493/2008) Dra. Vânia Margaret Flosi
Paschoalin; CAPES/PNPD (23038.007901/2010-81)
Dra. Ana Carolina da Silva Carvalho; and CNPq PhD
grant (140492/2013-5) Dr. Ricardo César Tavares
Introduction
Mycobacterium bovis is a bacteria belonging to the Mycobacterium tuberculosis complex
(MTC), which, in addition to causing tuberculosis in cattle and buffaloes (BTB), can cause dis-
ease in several species of mammals, including humans, thus being considered a zoonosis [
1,2].
BTB is a worldwide-distributed disease with striking prevalence in developing countries.
This disease has socio-economicimpacts by reducing livestock productivity due to early dis-
posal of high zootechnical value animals, reduction in weight gain of affected animals and loss
in the export of products from the cattle industry, mainly meat [
3,4].
Infection by M. bovis in humans is typically caused by the consumption of animal food
products contaminated by the bovine bacillus, usually unpasteurized milk and milk derivatives
[
5], leading to the development of tuberculosis in its extrapulmonary form [6]. Another route
for M. bovis infection in humans is through airborne transmission [
7,8]. These infections are
clinically and pathologically indistinguishable from tuberculosis (TB) caused by M. tuberculosis
[
9,6]. It is suspected that infections caused by M. bovis are responsible for more than 4000
cases among the 100,000 cases of human tuberculosis described annually in Brazil [
10,11].
However, according to the World Organization for Animal Health (OIE), the number of
human TB cases caused by M. bovis in Brazil cannot be estimated [
12], since bacteriological
culture followed by biochemical identification tests to diagnose whether the infective agent was
M. bovis or M. tuberculosis are not performed in most tuberculosis cases [
13].
Cattle raising is very important for the Brazilian economy. Currently, the cattle herd in the
country is over 212 million heads, and the Midwestern region, formed by the states of Mato
Grosso, Mato Grosso do Sul and Goiás, is the main cattle-producing region [14] and the largest
beef exporting region in the country [
15]. Although livestock sanitary risks could impact the
agribusiness on Brazilian economy, there is still a lack of updated data on the distribution and
prevalence of BTB in the country and in the different producing regions. The latest official
national prevalence data of the disease was in 2004, reporting a rate of 1.3% [
8]. On the other
hand, the estimated prevalence of the disease in the Midwest was of 0.37%, as described by
Roxo, in 2004 [16]. In a recent study, the estimated prevalence of BTB for the state of Mato
Grosso, which is part of the Midwest region, was estimated at 0.007% [
17]. It is believed that,
currently, the prevalence of BTB in the whole Midwestern region may be lower than that
described in 2004 [
16].
In order to reduce the prevalence and incidence of new BTB outbreaks in herds, to certify
properties as free or monitored for the disease, and to offer consumers lower health risk prod-
ucts, Brazil, the Ministry of Agriculture Livestock and Supply (MAP) launched the National
Program for Control and Eradication of Bovine Brucellosis and Tuberculosis (PNCEBT) [
8] in
2001, which was regulated in 2004. This animal health program recommends performing the
intradermal tuberculin test, followed by the slaughter of positive cattle, surveillance in slaugh-
terhouses, tracing the origin of the outbreak and sanitation, as established by the International
Organization for Animal Health [
18].
The molecular identification of strains involved in BTB infection may contribute to an
increased efficiency of disease control programs, since the identification of M. bovis genotypes
prevalent in a particular area, allows to track and control the occurrence of multiple foci of dis-
ease [
19,20], especially in areas with low prevalence of the disease, as is the case of the Brazilian
Midwestern region.
Spacer oligotyping (spoligotyping) and variable number tandem repeat (VNTR) are amply
used techniques in human tuberculosis epidemiological studies, as well as molecular typing of
MTC species, which includes M. bovis [
21]. When combined, spoligotyping and VNTR are
able to distinguish the bacteria lineages more effectively [
22,23,24], with a good cost/benefit
M. bovis Heterogenicity in Midwest Brazil
PLOS ONE | DOI:10.1371/journal.pone.0162459 September 15, 2016 2 / 16
Carvalho. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
relationship, due to speed, reproducibility and reliability of the performed genotyping
[
25,26,27,28].
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 capil-
lary electrophoresis [
34]. Each technique has its advantages and disadvantages that must be
considered when choosing which to implement in the laboratory. Spoligotyping in combina-
tion 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.
Materials and Methods
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 bio-
chemical [
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 (Invitro-
gen, CA, USA). DNA templates from M. bovis BCG and M. tuberculosis H37Rv were used as
positive controls in the spoligotyping and MIRU-VNTR assays.
Spoligotyping
The spoligotyping method was conducted as described by Kamerbeek et al. (1997) [
28]. Hybri-
disation of the PCR product to the spoligo-membrane was performed according to the manu-
facturer’s instructions (Ocimum Biosolutions, Telangana, IN). Bound fragments were detected
by chemiluminescence after incubation with peroxidase-labelled streptavidin (1:4000). Only
patterns with 100% similarity were considered as clusters. Those strains clustered by spoligo-
typing were analyzed by MIRU-VNTR to confirm their clonal relationships. M. bovis profiles
were compared to those available at the Mbovis.org website (
http://www.mbovis.org/) [36] and
SITVIT-WEB (
http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE/)databases.
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 recom-
mended 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) [
32], 1X KAPA2G Fast HotStar ReadMix
PCR Kit
1
(Kapabiosystems, MA, USA), 1.87 μl of DMSO [p.a.] and 2 μl of purified DNA
(about 20 ng) in a final volume of 20 μl. PCR assay conditions were 3 min at 95°C, followed by
30 cycles for 15 sec at 95°C, 15 sec at 59°C, 30 sec at 72°C and a final extension step at 72°C for
10 min.
M. bovis Heterogenicity in Midwest Brazil
PLOS ONE | DOI:10.1371/journal.pone.0162459 September 15, 2016 3 / 16
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 form-
amide (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].
The sample profiles were compared to those available at the database MIRU-VNTR plus
(
http://www.miru-vntrplus.org/MIRU/index.faces) and analyzed by BioNumerics software 6.6
(Applied Maths, Sint-Martens-Latem, BE).
Allelic and genotypic diversity calculations
The Hunter-Gaston discriminatory index (HGDI) [
38] was used to calculate the allelic diver-
sity 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 spoligo-
typing 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).
Results and Discussion
After the spoligotyping, the 37 M. bovis isolates were classified as (
Table 1) SB0121 (n = 14;
37.8%), SB0295 (n = 6, 16.2%), SB0140 (n = 6), SB0881 (n = 3, 8.1%), SB1144 (n = 2, 5.4%) and
SB1145 (n = 2). In addition, four strains (10.8%), SB0134, SB1050, SB1055 and SB1136, showed
orphan patterns. The geographic distribution of the spoligotypes is presented in
Fig 1.
The predominant spoligotype SB0121 was widespread in the three states of the Brazilian
Midwest, and has also been described as the most prevalent in other Brazilian regions, includ-
ing in the states of Rio Grande do Sul (92.9%), in the Southern region of the country [
19], São
Paulo (32.7%) [
39] and Minas Gerais (16.4%) [40], both in the Southeastern region, in the state
of Bahia (36%), in the Northeast, [
3] and in the state of Mato Grosso do Sul (30.7%), in the
Midwest [
20]. Outside Brazil, SB0121 has been described in the Netherlands [41], France
[
41,42], Italy [43], Belgium [41], Portugal [44], Spain [45], Algeria [46], South Africa [47],
Mexico [48,49] and Venezuela [49]. Interestingly, the SB0121 spoligotype has not yet described
in Argentina, a country that borders Brazil and where animal movement between the countries
frequently occurs [
49].
The second most frequent spoligotype, SB0295, found in Mato Grosso and Goiás has been
described in the states of São Paulo (35%) [
39] and, Bahia (14%) [3], consistent with the
national prevalence of 24% [
49]. The SB0295 spoligotype has also been described in Spain [50],
Portugal [44], France [42] and Mexico [51].
M. bovis Heterogenicity in Midwest Brazil
PLOS ONE | DOI:10.1371/journal.pone.0162459 September 15, 2016 4 / 16
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 Mid-
western 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
Table 1. Molecular characterization of the 37 M. bovis isolates by spoligotyping method.
Sample Spoligotype Spoligotype pattern
44 1100000101111110111101111000011111111100000 SB1145
45 1100000101111110111101111000011111111100000 SB1145
49 1101111101111110111101111000000111111100000 SB0881
52 1101111101111110111101111000000111111100000 SB0881
10 1101111101111110111101111000000111111100000 SB0881
35 1101111101111110111101111111111111111100000 SB0121
36 1101111101111110111101111111111111111100000 SB0121
11 1101111101111110111101111111111111111100000 SB0121
22 1101111101111110111101111111111111111100000 SB0121
23 1101111101111110111101111111111111111100000 SB0121
30 1101111101111110111101111111111111111100000 SB0121
37 1101111101111110111101111111111111111100000 SB0121
33 1101111101111110111101111111111111111100000 SB0121
39 1101111101111110111101111111111111111100000 SB0121
48 1101111101111110111101111111111111111100000 SB0121
17 1101111101111110111101111111111111111100000 SB0121
5 1101111101111110111101111111111111111100000 SB0121
4 1101111101111110111101111111111111111100000 SB0121
38 1101111101111110111101111111111111111100000 SB0121
15 1101111101111110111101111111111111110100000 SB0295
16 1101111101111110111101111111111111110100000 SB0295
13 1101111101111110111101111111111111110100000 SB0295
12 1101111101111110111101111111111111110100000 SB0295
25 1101111101111110111101111111111111110100000 SB0295
18 1101111101111110111101111111111111110100000 SB0295
1 1101111101111110111101111111100000001100000 SB1144
21 1101111101111110111101111111100000001100000 SB1144
19 1101101000001110111111111111111111111100000 SB0140
20 1101101000001110111111111111111111111100000 SB0140
27 1101101000001110111111111111111111111100000 SB0140
28 1101101000001110111111111111111111111100000 SB0140
46 1101101000001110111111111111111111111100000 SB0140
29 1101101000001110111111111111111111111100000 SB0140
14 1100011101111110111111111111111111111100000 SB0134
24 0000000000011110111111111111111111111100000 SB1136
3 1101111101111110111101111111100000111100000 SB1050
9 1100011101111110111111111111111111110100000 SB1055
M. bovis BCG 1101111101111110111111111111111111111100000 Reference strains
M. tuberculosis H37Rv 1111111111111111111001111111111100001111111 Reference strains
doi:10.1371/journal.pone.0162459.t001
M. bovis Heterogenicity in Midwest Brazil
PLOS ONE | DOI:10.1371/journal.pone.0162459 September 15, 2016 5 / 16