Ocorrência de schistosoma a nível mundial e perfil genético

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The role of the immunological background
of mice in the genetic variability of
Schistosomamansoni as detected by random
amplification of polymorphic DNA
I.L. Cossa-Moiane1*, T. Mendes2, T.M. Ferreira2, I. Mauricio2,
M. Calado2, A. Afonso2,3 and S. Belo2
1Laboratory of Molecular Parasitology, Departamento de Plataformas
Tecnolo´gicas, Instituto Nacional de Sau´de, Ministe´rio da Sau´de, Avenida
Eduardo Mondlane, 1008, PO BOX 264, Maputo, Mozambique: 2Medical
Parasitology Unit, Instituto de Higiene e Medicina Tropical/Unidade de
Parasitologia e Microbiologia Me´dicas, Universidade Nova de Lisboa,
Rua da Junqueira, 100, 1349-008 Lisboa, Portugal: 3Universidade de Sa˜o
Paulo (USP), Instituto de Quı´mica de Sa˜o Carlos, DQFM, Grupo de
Bioanalı´tica, Microfabricac¸a˜o e Separac¸o˜es, Sa˜o Carlos, Sa˜o Paulo, Brazil;
Universidade Federal de Sa˜o Carlos, Departamento de Morfologia e
Patologia, Sa˜o Carlos, Sa˜o Paulo, Brazil
(Received 12 February 2014; Accepted 31 May 2014)
Abstract
Schistosomiasis is a parasitic disease caused by flatworms of the genus
Schistosoma. Among the Schistosoma species known to infect humans, S. mansoni
is the most frequent cause of intestinal schistosomiasis in sub-Saharan Africa and
South America: the World Health Organization estimates that about 200,000
deaths per year result from schistosomiasis in sub-Saharan Africa alone. The
Schistosoma life cycle requires two different hosts: a snail as intermediate host
and a mammal as definitive host. People become infected when they come into
contact with water contaminated with free-living larvae (e.g. when swimming,
fishing, washing). Although S. mansoni has mechanisms for escaping the host
immune system, only a minority of infecting larvae develop into adults,
suggesting that strain selection occurs at the host level. To test this hypothesis,
we compared the Belo Horizonte (BH) strain of S. mansoni recovered from
definitive hosts with different immunological backgrounds using random
amplification of polymorphic DNA–polymerase chain reaction (RAPD-PCR).
Schistosoma mansoni DNA profiles of worms obtained from wild-type (CD1 and
C57BL/6J) and mutant (Ja182/2 and TGFbRIIdn) mice were analysed. Four
primers produced polymorphic profiles, which can therefore potentially be used
as reference biomarkers. All male worms were genetically distinct from females
isolated from the same host, with female worms showing more specific
fragments than males. Of the four host-derived schistosome populations, female
and male adults recovered from TGFbRIIdn mice showed RAPD-PCR profiles
*E-mail: idaleciacossa@yahoo.com.br
Journal of Helminthology, page 1 of 6 doi:10.1017/S0022149X14000492
q Cambridge University Press 2014
that were most similar to each other. Altogether, these data indicate that host
immunological backgrounds can influence the genetic diversity of parasite
populations.
Introduction
Schistosomiasis is a parasitic infection caused by
flatworms of the genus Schistosoma (Rey, 2010) and
among human parasitic diseases it is second only to
malaria as a cause ofmorbidity–mortality (Berriman et al.,
2009). It affects people living in tropical and subtropical
regions (Stothard et al., 1996) and the World Health
Organization estimates that more than 207 million people
are infected worldwide, most of them in poor commu-
nities and 85% living in Africa (Guerrant et al., 2011).
Perhaps 700 million people are at risk of infection due to
agricultural, domestic or recreational activities involving
contact with contaminated water, in which intermediate
hosts (freshwater snails) have released the infectious
larval forms of Schistosoma (King, 2011). Among the
described species, S. mansoni is the main cause of
intestinal schistosomiasis, which is endemic in Africa
and South America (Rey, 2010).
Schistosoma spp. use a wide range of strategies to evade
host immunity and thus facilitate their own development
and transmission (Karanja et al., 1997; He et al., 2001;
Cardoso et al., 2008); for example, infectivity, pathogen-
icity and immunogenicity can vary within the same
species, strain and sex of schistosome (Nino Incani et al.,
2001). The host immune response is also important for the
outcome of infection, with both invariant natural killer
T (iNKT) cells and the Th3 cytokine transforming growth
factor beta (TGF-b) affecting parasite development and
embryogenesis (Oliveira et al., 2012).
Molecular biology techniques such as random ampli-
fication of polymorphic DNA–polymerase chain reaction
(RAPD-PCR) have been used to detect genetic diversity in
isolates of S. mansoni from Brazil and other endemic areas
(Pillay & Pillay, 1994; King, 2011). Researchers
also identified genetic differences between S. mansoni
populations resistant or tolerant to praziquantel (PZQ),
oxamniquine (Oxa) and hycanthone (Hyc) by RAPD-PCR
(Tsai et al., 2000). Although some genetic differences are
likely to be neutral and simply reflect divergence time,
some polymorphisms may be markers of, or adaptations
to, the immune systems of different hosts (Berriman et al.,
2009). Such genetic polymorphisms may give the parasite
the capacity to colonize different hosts with distinct
immunological backgrounds (Tsai et al., 2000; Gentile &
Oliveira, 2008).
C57BL/6 mice are widely used in the development of
genetically modified mice (Smith, 2002). Mutant strains of
this lineage have been generated with different immune
environments (Wakao et al., 2007; Dwivedi et al., 2011),
such as Ja182/2 (deficient exclusively in iNKT cells) and
TGFbRIIdn (expressing a dominant-negative form of
TGFb receptor). Ja182/2 and TGFbRIIdn mice have been
used as models for autoimmune disease, anti-tumour
immune responses, inflammatory bowel disease (Mi et al.,
2011) and schistosome infections (Osman et al., 2006;
Mallevaey et al., 2007).
The aim of the present study was to evaluate genetic
differences between adults of the Belo Horizonte (BH)
strain of S. mansoni able to successfully infect mice with
distinct immunological backgrounds, and to determine
whether specific S. mansoni DNA markers are associated
with different host immune backgrounds.
Materials and methods
Parasitological procedures
TheBeloHorizonte (BH) strain ofS.mansoniwasobtained
from theMedical ParasitologyUnit of Instituto deHigiene e
Medicina Tropical (Lisbon). These clonal parasites are
originally from Belo Horizonte city, Brazil, and have been
continuouslymaintained for 20 years by successive passage
through CD1mice (Mus musculus) and Biomphalaria glabrata
snails. In this study, mouse lineages CD1 and C57BL/6J
(as controls), Ja182/2 and TGFbRIIdn, supplied by The
JacksonLaboratory (BarHarbor,Maine,USA),were infected
by exposing their tails to 50 cercariae ofS.mansoni in 50ml of
water for 2h. Infection occurred by dermal penetration.
Seven weeks post-infection, eggs were observed in faeces,
using the Telemann–Lima and Kato–Katz techniques
(WorldHealthOrganization, 2003).Oneweekafter infection
adult worms were recovered as described by Duvall &
DeWitt (1967). Recovered worms from each mouse strain
were divided into groups of males, females and pools of
males and females.
Molecular analysis
DNA was extracted from each pre-defined group of
worms using a modified cetyltrimethylammonium bro-
mide (CTAB)protocol (Stothard et al., 1996). Briefly,worms
were ground to a fine powder using liquid nitrogen, 600ml
of extraction buffer was added to each sample, then the
samplewasgroundagain and10ml proteinaseK (10mg/ml)
was added prior to incubation at 558C for 90min with
agitation.An equal volumeof chloroform : isoamyl alcohol
(24:1) was added and gently mixed 30–40 times, then
briefly centrifuged. To the supernatant was added 800ml
cold absolute ethanol and the mixture was centrifuged at
8000 g for 20min at room temperature. The pellet was
washed twice with 70% ethanol at 8000g for 15min. The
final pellet was air-dried and dissolved in Tris-EDTA (TE)
buffer (50ml). Finally ribonuclease (RNase, 10mg/ml)
was added, theDNA incubated at 378C for 30min and then
stored at 2208C until use.
For RAPD-PCR, ten-mer oligonucleotides described by
Tsai et al. (2000) were tested and used individually (i.e. a
single primer per reaction). For a final volume of 25ml,
50mM MgCl2, 10 pmol each primer, 25mg DNA and
milli-Q water (Merck Millipore, Lisbon, Portugal) were
added to Illustra PuReTaq Ready-to-Go PCR Beads
(GE Healthcare, Amersham, UK). Amplification reactions
were performed in a thermocycler (Mechatronic Systems
2 I.L. Cossa-Moiane et al.
GmbH , Wies, Austria) as described by Tsai et al. (2000).
Electrophoresis was carried out in 1.5% agarose gels,
20 cm in length, with 0.5% ethidium bromide (EtBr)
staining. All samples amplified with the same primer were
included in the same gel. DNA fragments were visualized
andphotographedunderUVlight (AlphamagerwHP,Alpha
Innotech, San Leandro, California, USA). The resulting
amplification patterns were digitized and analysed
automatically. A negative control and male and female
S. mansoni DNA from CD1 and C57BL/6J (as positive
controls) were used in all amplifications.
Primers that produced patterns with defined reprodu-
cible bands were selected. A data matrix was constructed
ofDNAfragmentsproducedby fourRAPDprimers (OPI-3,
OPI-7, OPI-12 and OPI-18) encoded as present (1) or
absent (0). A neighbour-joining dendrogram was built
using NEIGHBOR from a Modified Nei distance matrix
produced by the programme RESTDIST in PHYLIP
(Felsenstein, 1989), and analysed as restriction sites 20 bp
long, with a default transition/transversion ratio of 2. The
resulting tree was visualized and edited in MEGA5.10
(Tamura et al., 2011). No bootstrap analysis was
performed due to the small number of polymorphic
DNA fragments.
Results
Adult worms of the S. mansoni were collected from
infected CD1, C57BL/6J, Ja182/2 and TGFbRIIdn mice
andDNAwas prepared for RAPD-PCR analysis. Of the ten
primers tested byRAPD-PCR, all but one (OPI-5) produced
amplification products, which were between 200 and
2000bp in size. Four (OPI-3, OPI-7, OPI-12 and OPI-18) of
the nine successful primers gave polymorphic patterns.
For each class of host, the male worms recovered could
be distinguished from females by at least one of the
amplification patterns obtained with the four RAPD
primers (fig. 1). Sex-specific fragments were more often
found in females than males, and only worms from
TGFbRIIdn mice gave rise to sex-specific fragments for
both sexes. In the case of males recovered from CD1 and
TGFbRIIdn, two sex-specific fragments were identified
OPI-3 OPI-7
OPI-18OPI-12
M A B C D E M
M A B C D E M
2000 bp
1000 bp
500 bp
2000 bp
1000 bp
500 bp
2000 bp
1000 bp
500 bp
2000 bp
1000 bp
500 bp
M A B C D E M
M A B C D E M
Fig. 1. RAPD-PCR profiles using four primers OPI-3, OPI-7, OPI-12 and OPI-18 of adult worms of the BH strain of Schistosoma mansoni
from wild-type mouse strains CD1 (lanes A, B) and C57BL/6J (lane C) and mutant mouse strains Ja182/2 (lane D) and TGFbRIIdn
(lane E) with 2000 bp molecular weight markers (lanes M). Pools of female, male and mix of female and male worms are indicated from
left to right of each lane, with specific fragments arrowed (white).
RAPD-PCR polymorphisms in host-selected S. mansoni 3
(table 1). The 1400 bp fragment amplified with primer
OPI-18 was female-specific only for worms from C57BL/
6J and Ja182/2 hosts, while in other mouse strains both
male and female schistosomes gave rise to this fragment.
Primer OPI-7 amplified a fragment of 600 bp only from
DNA of males and females infecting TGFbRIIdn (table 1).
A neighbour-joining dendrogram, built from the
fragment data produced by the four polymorphic RAPD
primers (fig. 2), showed that S. mansoni females andmales
isolated from CD1 mice were more distantly related to
each other than males and females from other hosts. For
example, S. mansoni males and females from TGFbRIIdn
mice were less distinct than those fromCD1mice. Finally,
S. mansoni females isolated from C57BL/6J and Ja182/2
had indistinguishable profiles, whereas their respective
malesweredistinct andmore related to eachother (table 1).
Discussion
RAPD-PCRusesa singleprimer toproduceamplification
fragments that can be scored as present or absent (Avise,
2004). The technique allows the identification of genomic
regions of interestwithout needingprior information about
the organism being investigated (Welsh et al., 1991). As a
result, RAPD-PCR has been widely used for genetic
mapping, development of genetic markers linked to a
trait of interest, population studies, evolutionary genetics,
and plant and animal breeding (Bardakci, 2000). The ten
RAPD-PCRprimers used in this studyhadpreviously been
evaluated by Tsai et al. (2000) for the study of genetic
diversity in S. mansoni and, in our hands, polymorphic
profiles were observed using primers OPI-7, OPI-12 and
OPI-18.Male and female S. mansoniwormswere recovered
after infection of different mouse strains to investigate
whether the host influences the genetic diversity of the
parasite population. Interestingly, for each host tested, the
female schistosome recovered could be distinguished from
males according to their RAPD-PCR profiles. This suggests
some formof sex-linkedgenetic polymorphism, although it
was not possible to determine the gene(s) involved. Such
sex-linked polymorphism found in our study could be
explained by the peculiar biology of these parasites. In
the blood vessel of the definitive host, male and female
form a linked couple (Rey, 2010). Males accommodate
females in their gynecophoric canal and modulate
their development (Guerrant et al., 2011).
As well as sex-specific differences, worms isolated from
different mice also showed distinct RAPD-PCR profiles.
NKTcells have properties of both Tand NK cells, forming
a heterogeneous group that expresses an invariant T-cell
receptor (TCR) a-chain rearrangement; in mice this is
Va14Ja18, while in humans it is Va24Ja18. These iNKT
cells are important for the balance of Th1 and Th2
immune response in S.mansoni infection (Arosa et al., 2007;
Mallevaey et al., 2007). In our study, we used Ja182/2
mice, which do not have iNKT cells; therefore this is
likely to affect the populations of schistosomes. In the
TGFbRIIdn background, in which a dominant-negative
form of the TGF-b receptor is expressed, both male and
female RAPD-PCR profiles were quite distinct from each
other and from those of other strains, although each
S. mansonimale–female pair belonged to a single branch.
This suggests that TGF-b deficiency selects quite specific
subsets of worms and, strikingly, genetically distinct male
and female populations, consistent with selectively
advantageous genotypes in each sex.
The effect of human TGF-b (hTGF-b) expression on
adult profiles of S. mansoni has been shown to promote
Table 1. The size of specific RAPD fragments in Schistosoma mansoni populations relative to mouse strain and primer.
Host mouse strain
CD1 C57BL/6J Ja182/2 TGFbRIIdn
Total no. specific
fragmentsPrimer Female Male Female Male Female Male Female Male
OPI-3 – – – – – – – 1100 bp 1
OPI-7 – 690 bp – – – – 900 bp 600bp 3
– 650 bp – – – – 600 bp – 2
OPI-12 – – – – 1500bp – – 1300 bp 2
OPI-18 – – 1400bp – 1400bp – – – 2
Total no. specific fragments 0 2 1 0 2 0 2 3 10
CD1-
CD1-
TGFβRIIdn-
TGFβRIIdn-
Jα18–/– -
Jα18–/–-
C57BL/6J-
0.001
C57BL/6J-
Fig. 2. Dendrogram analysis of RAPD pofiles. Neighbour-joining
dendrogram built by NEIGHBOR from a Modified Nei distance
matrix produced by the programme RESTDIST in PHYLIP
(Felsenstein, 1989), with 57 characters in total from four RAPD
primers analysed as restriction sites20 bp long with a
transition/transversion ratio of 2. The tree was visualized and
edited in MEGA5.10 (Tamura et al., 2011).
4 I.L. Cossa-Moiane et al.
genetic variations in helminths (Oliveira et al., 2012). In
fact, host gene expression has been associated with the
morphology, development and life cycle of S. mansoni
(Arosa et al., 2007) and that could reflect on subtle genetic
changes that we have observed in our study. Thus, we
could speculate that the immunological background of
the host might select genetic variation, since TGF-b
can acts a regulator of chronic helminthic infection
(Ziegler, 2006).
In summary, we have demonstrated that RAPD-PCR is
a useful tool for assessing genetic differences between
experimental populations of S. mansoni and could lead to
the discovery of specific host interaction markers. The
molecular mechanisms involved in host–parasite inter-
action are complex and imply bilateral cross-talk, with
signals in both directions (Arosa et al., 2007) giving rise to
distinct genetic variants. However, further experiments
are needed to elucidate the role of the host environment in
the evolution of S. mansoni populations and to better
understand the molecular mechanisms involved in
infection. For example, microsatellite and sequencing of
major specific fragments between worms from hosts with
different immunological backgrounds could be useful
for better understanding of the molecular mechanisms
involved in murine schistosomiasis assays.
Acknowledgements
The authors are very grateful to Dr Milton Moraes and
Dr Tufa´ria Mussa´ for their assistance and technical
support in the writing of this article.
Financial support
This work was supported by the Foundation for
Science and Technology (Pest-OE/SAU/UI0074/2011),
Lisbon, Portugal.
Conflict of interest
None.
Ethical standards
The study was approved by the Portuguese Commis-
sion on Ethics and Animal Welfare (Ref. 0421/2013), and
all animals were treated and maintained in accordance
with National and European legislation on the protection
of animals used for scientific purposes (2010/63/UE
adopted on 22 September 2010).
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