RESEARCH ARTICLE
Mixed infection of Leishma
r
o
2, L
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Ferreira et al. BMC Veterinary Research  (2015) 11:71 
DOI 10.1186/s12917-015-0392-yspread to many other areas, including the municipalityBrasil
Full list of author information is available at the end of the articleBackground
The epidemiology of leishmaniases in the New World
(NW) is very complex due to the participation of several
species of Leishmania, phlebotomine vectors and mam-
malian hosts. NW visceral leshmaniasis (VL) is endemic
or sporadic and is caused by Leishmania (Leishmania)
infantum (syn. L. (L.) chagasi). Children under 10 years
of age are the main affected population, but adults can also
be frequently affected in foci of recent introductions.
Several phylogenetically distinct species of Leishmania
are responsible for the forms of cutaneous leishmaniasis
(CL), from localized cutaneous lesions to the destructive
mucosal affliction [1].
Cutaneous leishmaniasis is widespread in Brazil, which
accounts for most CL cases in South America with an
estimated average incidence of 100.5 cases per 100,000
inhabitants. Until the 1950s most of the cases of CL in
Brazil were concentrated in the states of São Paulo,
Paraná, Minas Gerais, Ceará and Pernambuco, and asso-
ciated with deforestation and new human settlements.
Due to the expansion of these activities the disease has
* Correspondence: ecferreira@fiocruz.br
1Fiocruz Mato Grosso do Sul, Fundação Oswaldo Cruz, Campo Grande, MS,Abstract
Background: In Brazil Leishmania braziliensis and L. infantum are the principal species responsible for cutaneous
and visceral leishmaniases, respectively. Domestic dogs are the main reservoirs of visceral leishmaniasis, while
rodents and marsupials are the main reservoirs for cutaneous leishmaniasis. It has also been suggested that dogs
could play a role in transmission of cutaneous leishmaniasis. The identification of the species of Leishmania, the
reservoirs, and the vectors involved in each particular transmission cycle is critical for the establishment of control
activities. Belo Horizonte has emerged as an endemic region for leishmaniases, however, epidemiological studies
assessing the contribution of wild reservoirs to transmission are scarce in the area. The aim of this study was to
investigate Leishmania spp. infection in possible reservoirs of an urbanized area.
Results: A high rate of infection was found in small mammals (64.9%) and dogs (DG1 30.4% and DG2 48.6%). The
presence of L. infantum and L. braziliensis was detected in small mammals and dogs, and mixed infections by both
species were detected in rodents which, to the best of our knowledge, is the first description of this phenomenon
in an urban area. Additionally, L. amazonensis was detected in the canine samples.
Conclusion: The possible role of these animals as a source of infection of the vector of each species of Leishmania
identified should not be overlooked and should be taken into account in future control activities. The results of
mixed infection by L. braziliensis and L. infantum in cosmopolitan rodents as M. musculus and R. rattus, may have
important implications in the context of the control of leishmaniasis in urban areas, especially when considering
that these rodents live in close relationship with human dwellings, especially those in more precarious conditions.
Keywords: Leishmania, Hosts, Reservoir, Small mammals, Dogs, Mixed-infectionsLeishmania braziliensis in
urban area of the New W
Eduardo de Castro Ferreira1*, Israel Cruz2, Carmen Cañavate
Filipe A M Madeira4, Sofia Alves Nogueira Valério5, Heito
and Célia Maria Ferreira Gontijo7© 2015 Ferreira et al.; licensee BioMed Central
Commons Attribution License (http://creativec
reproduction in any medium, provided the or
Dedication waiver (http://creativecommons.or
unless otherwise stated.Open Access
nia infantum and
odents from endemic
rld
utiana Amaral de Melo3, Agnes Antônia Sampaio Pereira7,
orais Cunha4, Adriano Pereira Paglia6. This is an Open Access article distributed under the terms of the Creative
ommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
iginal work is properly credited. The Creative Commons Public Domain
g/publicdomain/zero/1.0/) applies to the data made available in this article,
Study design animals and samples
The number of samples was defined by estimating the
proportion of the population with a confidence level of
95%, precision of 0.1 and prevalence of 50%. This value
was estimated without any previous data and consider-
ing the prevalence of 50% gives the largest “n” possible.
The calculated n was 100, both for dogs and for small
mammals.
The project obtained permission from the Ministry of
Environment Agency in Brazil, the Brazilian Institute for
Environment (IBAMA), for capture and euthanasia of
small mammals.
Ferreira et al. BMC Veterinary Research  (2015) 11:71 Page 2 of 7of Belo Horizonte, the capital of the state of Minas Gerais
[2]. VL is also a serious public health problem in Brazil,
with escalating incidence (1.85/100,000 inhabitants) and
broadening geographical extension; northeastern states
are particularly affected. Currently, VL is strongly linked
to urban settlements in Brazil, where human made
changes as well as adaptive changes in the behavior of
reservoirs and vectors contribute to its prevalence. Thus,
since 1980 several urban VL epidemics have taken place
in Brazilian cities, including Belo Horizonte [3,4].
Several domestic, synanthropic, and wild animals have
been found infected by different species of Leishmania
in urban areas, thus the identification of sources of infec-
tion and the epidemiology of each clinical form of leish-
maniasis becomes a complex matter [1,5-11]. Throughout
the last decade, the detection of leishmanial DNA has
been shown to be a useful approach in detecting
Leishmania infection in dogs and other mammals in
urban and sylvatic environments in Brazil, and particu-
larly in the state of Minas Gerais [12-18].
Both CL and VL have been known to be endemic in
Belo Horizonte for a long time [19,20]. This is a heavily
urbanized and densely inhabited area, where transmis-
sion of leishmaniases occurs in practically all quarters of
the city, resembling an urban transmission pattern
already described [2,3,21,22].
In this study we aimed to use nested polymerase chain
reaction (PCR) to target the SSUrRNA gene [23] in order
to assess the presence of Leishmania infection in periph-
eral blood and different tissues obtained from dogs and
small mammals from the Municipality of Belo Horizonte.
The PCR method used has been validated and applied in
previous studies assessing Leishmania infection in both
humans and dogs [24,25]. In combination with DNA
sequencing of positive PCR products we attempted to
identify the Leishmania species complexes infecting the
study animals. We believe that studies such as ours
will contribute to an improved understanding of the
epidemiology of the leishmaniases and will help to
establish further control activities.
Methods
Study location
The study was performed from November 2007 to
March 2008 in the Municipality of Belo Horizonte (capital
of Minas Gerais State, Brazil). The city is built on several
hills, is completely surrounded by mountains, and has
several large parks in its immediate surroundings. Belo
Horizonte has a global surface area of 331Km2, and is
characterized by a subtropical climate with annual pre-
cipitation of 1491 mm and average temperature of 22°C
(11°C—31°C). The human population of Belo Horizonte
is 2,375,151 inhabitants [26] and is undergoing intense
urbanization.The points of collecting samples of dogs and small
mammals in domiciliary and peridomestic areas were
chosen considering the presence of human cases of VL
and/or LT in the household, or close to it, in the three
years preceding the start of research.
The study was based on a sample of 189 animals
originating from two sources: i) 97 small mammals (62
rodents and 35 marsupials) captured with Tomahawk
traps from peri-domestic habitats (20 houses, each one
with two traps) and from forested areas in early succes-
sion around different households in Belo Horizonte
(three tracks, each containing 15 traps separated by a
distance of about 20 meters) (Table 1). The sampling
effort, at the end of the six campaigns, was 1200 traps
in peridomestic area and 1350 traps in the forested
area, adding up to a total of 2550 traps during the
period of study ; and ii) 92 domestic dogs (with owners)
from the same households from which small mammals
were captured (hereinafter dog group-1, DG1).
An additional set of dog samples was included in this
study (hereinafter dog group-2, DG2). This group was
made up of DNA extracted from peripheral blood, bone
marrow and ear skin of 70 dogs obtained in study loca-
tion during a previous survey [27] , to evaluate whether
there were changes in the profile of infection in dogs
from the same area, in both periods studied, applying
the same methodology on samples of the two groups.
Table 1 Number of individuals of small mammal species
captured in each habitat between June 2006 to November
2007 in Belo Horizonte, Minas Gerais, Brasil
Order Species Forest Peridomestic Total
Rodentia Mus musculus 4 20 24
Rattus rattus 8 11 19
Rattus norvegicus 1 8 9
Necromys lasiurus* 5 0 5
Cerradomys subflavus* 5 0 5
Didelphimorphia Didelphis albiventris* 31 3 34
Didelphis aurita* 1 0 1Total 55 42 97
*Native species.
and the automated ABI PRISM 377 DNA sequencer
(Applied Biosystems). Sequences thus obtained were
analyzed and edited using Lasergene® sequence analysis
software (DNASTAR). The edited sequences were com-
pared with those deposited at GenBank.
Statistical analysis
The differences of positive frequencies were analyzed by
Ferreira et al. BMC Veterinary Research  (2015) 11:71 Page 3 of 7Small mammals were anaesthetized and sacrificed, and
then samples from tail skin, ear skin, liver, spleen, and
bone marrow were collected. Peripheral blood samples
were taken from dogs. Peripheral blood and bone
marrow samples were preserved in EDTA tubes at −20°
C whereas the other tissue samples were stored in 70%
ethanol at −20°C until analysis.
Informed consent was obtained from each dog owner
before sampling. The procedures employed for the col-
lection of clinical material from dogs and small mam-
mals were carried out according to the Ethical Principles
in Animal Experimentation and the project was licensed
by the Ethics Commission on the Use of Animals/FIO-
CRUZ under reference number P0119-02.
DNA extraction
The Genomic Prep Cells and Tissues DNA Isolation Kit®
(GE Healthcare) was used for DNA extraction from skin,
liver, and spleen samples and the Column Chromatog-
raphy-GFX Genomic DNA Blood Purification System®
(GE Healthcare) was used for peripheral blood and bone
marrow samples. In both cases the instructions provided
by the manufacturer were followed.
Detection of Leishmania DNA
Detection of Leishmania DNA was done by means of
Leishmania nested-PCR (LnPCR) targeting the SSUrRNA
gene, as described elsewhere [24]. This protocol is specific
to the genus Leishmania and uses the primer pair R221
(5’-GGT TCCTTT CCT GAT TTA CG-3’) and R332 (5’-
GGC CGGTAA AGG CCG AAT AG-3’) in the first reac-
tion, and R223 (5’-TCC CAT CGC AAC CTC GGT T-3’)
and R333 (5’-AAA GCG GGC GCG GTG CTG-3’) in
the nested reaction. The PCR reagents (Biotools B&M
Labs, SA) and the thermal cycler (GenAmp PCR System
9800, Applied Biosystems) were used following the con-
ditions and the cycling program previously described
[24]. Every assay included DNA extraction negative and
positive controls and PCR negative and positive controls.
The presence of the final specific positive LnPCR product
(353 bp) was assessed by 1.5% agarose gel electrophoresis,
ethidium bromide staining and UV visualization. The
detection limit observed was equivalent to 1 and 10
parasites/uL in culture samples of reference strains of
L. infantum and L. braziliensis, respectively.
DNA sequencing
For species identification, all LnPCR positive products
were excised from the gel and purified using the QIA-
quick Gel Extraction Kit (QIAGEN) following the proto-
col provided by the manufacturer. Direct sequencing of
the purified LnPCR positive products was performed
with forward and reverse primers using the Big-Dye
Terminator Cycle Sequencing Ready Reaction Kit V3.1chi-square test (x2) and for multiple comparisons was
used the Bonferroni method. The level of significance
was 5%.
Results
We captured a total of five species of rodents and two
species of marsupials in both habitats (Table 1). Native
wild species were captured mostly in forested areas
while in the peri-domestic areas all species captured, ex-
cept the white-eared opossum (Didelphis albiventris),
were exotic and synanthropic.
After LnPCR analysis of the different biological sam-
ples any animal presenting an LnPCR positive result
(Figure 1) in at least one of the biological samples tested
was considered infected. Small mammals (N = 97) had
the highest infection rate (47.4–83.3%), which varied ac-
cording to species (details in Table 2). Among dogs,
DG1 (N = 92) showed an infection rate of 30.4%, while
DG-2 (N = 70) had an infection rate of 48.6% (Table 2).
Among small mammals, samples from liver, spleen and
tail skin showed a higher infection rate (22.4–33.7%)
than samples taken from ear skin and bone marrow
(5.2–10.2%). No significant differences were observed in
the infection rate of the different samples taken from
DG2 (Table 3).
DNA sequence analysis of the amplified fragment of
the SSUrRNA gene indicated that species from the L.
brazilensis complex were infecting 54%, 82.1% and 5.9%
of the LnPCR positive small mammals, DG-1 and DG-2,
respectively. Infection by species of the L. donovani
complex was determined in 12.7%, 17.9% and 82.3%,
of the LnPCR positive small mammals, DG1 and DG2,
respectively. When different Leishmania species com-
plexes were identified in different biological samples
from the same animal the infection was considered
Figure 1 Agarose gel electrophosis of the amplication products
from LnPCR for the SSUrRNA gene. Samples: MM:100 bpmolecular
size marker; NC: Negative Control; 01 to 10: amplifield DNA from
mammal samples; PC: Positive control-L.braziliensis (MOHM/BR/75/
M2903)DNA.
complex in a sample of Rattus norvegicus. This method-
ology, however, only allows identification to the level of
species complex and further analysis using other DNA
targets, such as the Hsp70 gene, would be necessary to
allow identification to the species level [29].
The present study also identified, for the first time,
mixed infection by the L. donovani species complex and
the L. braziliensis species complex in three small mam-
mals (two M. musculus and one R. rattus) captured in
peri-domestic habitats of three different households of
Belo Horizonte. The overlapping of transmission cycles
of different species of Leishmania in a given area can
lead to the occurrence of mixed infections, as has been
Table 2 Positivity of LnPCR in samples of rodents,
marsupials and dogs from Belo Horizonte, Minas Gerais,
Brazil
Host specie Sample LnPCR
M. musculus ** 83.3%
R. rattus ** 47.4%
R. norvegicus ** 66.7%
N. lasiurus ** 60%
C. subflavus ** 80%
D. albiventris ** 61.8%
D. aurita ** 0%
C. familiaris DG1 Blood 30.4%
Ferreira et al. BMC Veterinary Research  (2015) 11:71 Page 4 of 7mixed; mixed infection by species of the L. donovani
complex and L. braziliensis complex were detected in
4.7% of the LnPCR positive small mammals (two Mus
musculus, and one Rattus rattus). Infection by L. ama-
zonensis was observed in 5.9% of the LnPCR positive
dogs of DG2. Due to weak amplification it was not pos-
sible to obtain accurate DNA sequences for species
complex identification in 28.6% and 5.9% of the LnPCR
positive small mammals and dogs of DG2, respectively.
Details on the species complex identification for each
animal species are provided in Table 4.
Discussion
The present study represents the first time that PCR tar-
geting the SSUrRNA gene and further DNA sequencing
has been used in an epidemiological study on samples
C. familiaris DG2 ** 48.6%
**Positivity in at least one evaluated tissue, including blood, ear skin, and
bone marrow for C. familiaris (2) and ear skin, tail skin, liver, spleen, and bone
marrow for the other hosts.from both domestic dogs and small mammals. Further-
more, this study adds to the previous work by Marcelino
and colleagues [28], who applied the same methodology
to confirm infection by the L. braziliensis species
Table 3 Positivity of Leishmania sp. specific PCRs performed o
and dogs
Host specie Ear skin Tail skin Bo
M. musculus 12.5% 17.4% 5%
R. rattus 15.8% 5.3% 5.
R. norvegicus 33.3% 33.3% 0%
N. lasiurus 0% 0% 0%
C. subflavus 0% 0% 20
D. albiventris 3% 38.2% 6.
D. aurita 0% 0% 0%
C. familiaris DG1 NR NR NR
C. familiaris DG2 25.7% NR 32
NR_Unrealised.observed by other authors [30-33]. This is an astonishing
finding since these two species of rodents are commonly
found in urban areas close to domestic habitats and in
association with inadequate hygiene and environmental
disorganization.
In epidemiological studies in areas of occurrence of
different species of Leishmania, the collection of bio-
logical samples from different tissues increases not only
the probability of detecting mixed infections [30], but
also the chance of detecting the parasite itself, as shown
in Table 3.
Another fact that caught our attention was the pres-
ence of domestic dogs infected by Leishmania belonging
to the L. mexicana complex. This finding, indicates that
sympatric transmission of L. amazonensis species of the
L. braziliensis and L. donovani complexes is occurring.
The species of Leishmania most prevalent in small
mammals, both in the forested and peri-domestic areas,
was L. braziliensis followed by L. infantum. Although
none of the small mammals were found to be infected
by L. amazonensis, the possibility should not be entirely
ruled out since it was not possible to identify the etio-
logic agent of 29.7% of the small mammals that were
infected. This is especially true if we consider that this
species of Leishmania is encountered in dogs [34], and
n DNA extracted from different tissues of small mammals
ne marrow Liver Spleen Blood
56.5% 60.9% NR
3% 26.3% 5.3% NR
55.5% 0% NR
60% 60% NR
% 60% 40% NR
1% 14.7% 20.6% NR
0% 0% NR
NR NR 31.5%.9% NR NR 21.4%
he
ple
L
1
0
1
0
2
1
1
8
ar s
i),
Ferreira et al. BMC Veterinary Research  (2015) 11:71 Page 5 of 7other authors have reported the detection of infection by
this species in small mammals from different regions of
Brazil [14,35,36].
The identification of the parasites present in infected
dogs and small mammals has revealed species belonging
to the Leishmania braziliensis, Leishmania mexicana,
and Leishmania donovani complexes. This, combined
with the results of Saraiva and colleagues [37] who de-
tected the presence of these species in phlebotomines,
confirms the occurrence of an active transmission cycle
of these different species in Belo Horizonte.
In captures of sandflies taken during the same period
of DG2 samples collection, 68% of specimens were L.
longipalpis [38]. Moreover, in a survey conducted in the
same area and period that the DG1 and small mammal
samples were collected, 75% of the sandfly specimens
captured were L. whitmani [39]. The above mentioned,
prevalence of L. infantum in DG2 and L. braziliensis in
DG1 and small mammals are understandable given the
predominance of the respective vectors in the different
periods.
Most of the necessary conditions for incriminating a
Table 4 Identification of Leishmania by the sequencing of t
Host species (n) Sample Leishmania Com
L.b
M. musculus (20) ** 55%
R. rattus (09) ** 44.4%
R. norvegicus (06) ** 66.6%
N. lasiurus (03) ** 100%
C. subflavus (04) ** 50%
D. albiventris (21) ** 47.6%
C. familiaris DG1 (28) Blood 82%
C. familiaris DG2 (34) ** 5.9%
**Identification carried out in at least one positive tissue by the PCR, including e
ear skin, and bone marrow for DG2. L. b (Leishmania braziliensis), L. d (L. donovanspecies as a reservoir of Leishmania [11], was observed
in the dogs and small mammals sampled in our study.
Among commensal synanthropic species, the rat
(R. norvegicus), the roof rat (R. rattus), and the mouse
(M. musculus) are particularly important because they
have a cosmopolitan distribution and are responsible
for most of the economic losses and health caused to
man [40]. The high rates of infection detected in these
species, as well as the possibility of harboring different
species of Leishmania, coupled with the proliferation of
these animals in urban areas suggest the possibility that
they are contributing to the maintenance of the transmis-
sion cycles of Leishmania sp. Moreover, these synanthro-
pic species were also trapped in the forested areas. Freitas
and colleagues [41] also suggest that M. musculus parti-
cipates in the transmission cycle of L. braziliensis in an
endemic area of the state of Mato Grosso, Brazil.With respect to the white-eared opossum D. albiventris,
this species showed a high rate of infection in tail skin
(38.2%) with parasites characterized as L. braziliensis or
L. infantum, which corroborates the findings of numerous
authors [5,16,18,36,42,43]. Although most specimens
were captured in the forested area, D. albiventris has a
large home range and highly synanthropic habits, which
could make them a link between wild and urban trans-
mission cycles.
The presence of dogs infected with L. braziliensis and
L. infantum in a city like Belo Horizonte is consistent
with what has been described by other authors in LT
and LV endemic areas [3,44,45]. In addition, the high
percentage of dogs infected by L. braziliensis in an area
with LV transmission should be evaluated by other
methods, besides the serological diagnosis, to perform
euthanasia of seropositive dogs, as recommended by the
Ministry of Health of Brazil for LV control [46]; it is
known the possibility of cross-reactions by serological
tests used in Brazil for diagnosis of dogs infected by
L. braziliensis [27].
The present study shows that three different leishman-
LnPCR product in samples of small mammals and dogs
x
.d L.b + L.d L.m Leishmania sp
0% 10% 0% 25%
% 11.2% 0% 44.4%
6.7% 0% 0% 16.7%
% 0% 0% 0%
5% 0% 0% 25%
9% 0% 0% 33.4%
8% 0% 0% 0%
2.3% 0% 5.9% 5.9%
kin, tail skin, liver, spleen, and bone marrow for the small mammals and blood,
L.m (L.mexicana).ial entities (L. donovani species complex, L. braziliensis
species complex, and L. mexicana species complex) are
present as etiological agents in the infection of domestic
dogs from the Belo Horizonte, which is considered an
endemic area for VL and CL. Additionally, small mammals
in peri-domestic and forested areas are also infected by
parasites of the L. donovani species complex or the
L. braziliensis species complex.
Conclusions
Mixed infection by L. braziliensis and L. infantum in
cosmopolitan rodents as M. musculus and R. rattus, may
have important implications in the context of the
control of leishmaniasis in urban areas, especially when
considering that these rodents live in close relationship
with human dwellings, especially those in more precar-
ious conditions. The possible role of these animals as a
Ferreira et al. BMC Veterinary Research  (2015) 11:71 Page 6 of 7source of infection for the vector of each Leishmania sp.
identified in Belo Horizonte should not be overlooked
and should be taken into account in future control
activities. Furthermore, studies of the hosts of Leish-
mania spp., the potential reservoirs of these parasites,
should be encouraged in several endemic areas of the
disease in order to understand the real role of the
different host species in the complex eco-epidemiology
of leishmaniasis.
Abbreviations
NW: New World; L.: Leishmania; VL: Visceral leishmaniasis; CL: Cutaneous
leishmaniasis; PCR: Polymerase chain reaction; DG1: Dog group 1; DG2: Dog
group 2; LnPCR: Leishmania nested PCR.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CMFG and ECF conceived the study; IC and CC assisted with study design;
APP determined the trapping locations for small mammals; ECF, LAM, HMC,
FAMM, SANV and APP conducted field sampling; HMC, FAMM and APP
performed small mammal species identification; ECF LAM, AASP carried out
DNA extraction; ECF, IC and AASP carried out PCR, DNA sequencing, and
analyses of sequences thus generated; ECF, CMFG, AASP and IC drafted the
manuscript; All authors read and approved the final version of the
manuscript.
Acknowledgments
The authors wish to thank the Fundação de Amparo à Pesquisa do Estado
de Minas Gerais (FAPEMIG), the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (Capes), the Fundação Oswaldo Cruz (FIOCRUZ)
and European Community (EC - LeishEpiNetSA Consortium) for financially
supporting the study. We are also grateful to the Centro de Controle de
Zoonoses da Prefeitura de Belo Horizonte for assistance and logistic support,
and to Zoraida del Carmen Fernandez Grillo for assistance in revising the text
in English.
Author details
1Fiocruz Mato Grosso do Sul, Fundação Oswaldo Cruz, Campo Grande, MS,
Brasil. 2WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología,
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madri,
Espanha. 3Serviço de Biologia Molecular e Bioinformática, Diretoria de
Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG,
Brasil. 4Sete Soluções e Tecnologias Ambientais, Belo Horizonte, MG, Brasil.
5Regional Nordeste, Secretaria de Saúde de Belo Horizonte, Belo Horizonte,
MG, Brasil. 6Departamento de Biologia Geral, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil. 7Grupo de
Estudos em Leishmanioses, Centro de Pesquisas René Rachou, Fundação
Oswaldo Cruz, Belo Horizonte, MG, Brasil.
Received: 27 October 2014 Accepted: 11 March 2015
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