- Open Access
Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis
© Cunha et al.; licensee BioMed Central Ltd. 2013
- Received: 15 February 2013
- Accepted: 17 April 2013
- Published: 26 April 2013
Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis.
In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis.
Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.
Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.
- Leishmania infantum
- Clinical isolates
- Visceral leishmaniasis
- Molecular typing
Parasites from the Leishmania genus are trypanosomatid protozoans responsible for a group of diseases with a broad range of clinical manifestations collectively known as leishmaniasis (reviewed in [1–3]). The emergence of leishmaniasis as an opportunistic infection in HIV+ patients in areas where both pathogens are endemic  has generated new interest in leishmaniasis.
It is well known that species such as L. major and L. mexicana are usually exclusively dermotropic, while L. infantum and L. donovani are responsible for both cutaneous and visceral leishmaniasis . Apart from a general species-specific organ tropism of Leishmania, intraspecies intrinsic characteristics are also a relevant factor to consider. According to Maia et al. , dermotropic and viscerotropic L. infantum strains modulate the sand fly biting time on the host leading to the delivery, respectively, of a high or low dose of metacyclic promastigotes into the skin which will impact on the parasite tropism and manifestation of the disease. Even strains belonging to the same zymodeme have been associated to differential infectivity .
In experimental infections, however, another parasite-related feature is of major importance. In vitro cultivation of Leishmania is a subject open to wide variation between laboratories, making the comparison of similar experiments ambiguous. Depending on the culture medium (Santarém, N. and Cunha, J., submitted results and ), the duration of the culture  and the number of axenic passages performed , the promastigotes generated will be differentially enriched in metacyclic forms , which will condition the success of the infection. Nonetheless, the genetics and the immune status of the host play a similarly important role in the tropism and severity of the disease . In the murine models, L. major was only found in the infection site of the resistant C57BL/6 mice after subcutaneous injection, whereas the same experimental protocol followed in the susceptible BALB/c strain allowed visceralization . Also, high and low infective strains maintained their profile (visceralizing or regulatory, respectively) in BALB/c and C.B-17 SCID mice, although with higher parasite loads in the T and B cell-dysfunctional SCID animals .
The analysis of HIV/Leishmania-coinfected human patients brought important insights into the role of the immune system on the severity of the disease. On the one hand the visceralization of dermotropic strains is frequently observed in HIV/Leishmania-coinfections , as well as the regular presence of amastigotes in uncommon locations such as the lungs or the intestine . On the other, the appearance of unique Leishmania zymodemes in HIV+ patients has been reported, which may be indicative of circulating strains normally associated with asymptomatic disease in immunocompetent patients [13, 15]. Some studies have shown that strains originating from HIV+ patients have low infectivity, which explains its appearance only in immunocompromised individuals [7, 16]. On the contrary, three distinct infective profiles were attributed to strains responsible for CL or VL (from immunocompetent or HIV+ patients) and no correlation was made according to the origin of the isolate .
In this study, we have focused on four different L. infantum strains isolated from patients with CL, VL and HIV/Leishmania coinfections. We characterized these strains according to molecular, biological and infectivity characteristics. We standardized the in vitro culture to avoid any biased infectivity that was evaluated with macrophage and mouse models. We have studied the distribution of the strains in acute and chronic infection by qPCR assessing the parasite load in spleen, liver, bone marrow, blood and lymph nodes and correlated differences in infectivity with major findings on the molecular typing.
Four L. infantum strains isolated from patients in the Mediterranean basin and Portugal were used in this study. MHOM/MA/67/ITMAP-263 (ST) is a cloned line derived from a patient with visceral leishmaniasis [9, 18] that was used as an internal and comparative control in all the experiments performed. HL strain (MHOM/PT/2009/LLM-1708) was isolated from an immunocompetent patient with cutaneous leishmaniasis. Briefly, a skin biopsy was dissociated in a cell strainer to isolate the cells and was then transferred into culture in RPMI. E390M (MHOM/ES/99/LLM-855) and Bibiano (MHOM/ES/01/LLM-1083) were isolated from bone marrow aspirates of HIV/Leishmania-coinfected patients, this second one being responsible for recurrent relapses of leishmaniasis. The bone marrow samples were cultivated in NNN medium at 26–27°C, until the expansion of the promastigotes. ST, HL and Bibiano strains have been characterized by multilocus enzyme electrophoresis (MLEE) as MON-1 zymodeme, while E390M is MON-284 (electrophoretic mobilities for malate dehydrogenase (MDH) and glucose-phosphate isomerase (GPI) were determined to be of 104 and 105, respectively, in relation to MON-1 zymodeme ).
L. infantum isolates were subjected to molecular typing by targeting four different regions of the Leishmania genome. Leishmania DNA was extracted by phenol/chloroform as described below in more detail and samples were adjusted to a final concentration of 10 ng/μL after measuring DNA content with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). A volume of 5 μL of each sample was used in further PCRs. PCR products were run on 2% agarose gels stained with ethidium bromide and visualized under UV light. Then they were excised from agarose gels and purified using the QIAquick Gel Extraction Kit (QIAGEN). First, the species status of the isolates was confirmed by DNA sequencing of the heat-shock protein 70 (hsp70) gene . Further subtyping was performed by sequence analysis of the ribosomal internal transcribed spacer 1 (ITS1) and 2 (ITS2)  and the hydrophilic acylated surface protein B (hasp B) or k26 gene . The Big-Dye Terminator Cycle Sequencing Ready Reaction Kit V3.1 and the automated ABI PRISM 377 DNA sequencer (Applied Biosystems) were used for direct sequencing of the k26, ITS1 and ITS2 PCR products that was performed with the corresponding forward and reverse primers; internal primers for sequencing were also used for the hsp70 PCR product, as described by Fraga et al. . The obtained sequences were analyzed and edited using the software BioEdit Sequence Alignment Editor, version 188.8.131.52 (Ibis Biosciences) . ClustalW multiple alignment algorithm tool and manual adjustment were used for comparison of the resulting sequences with the respective published sequences. The hsp70 sequences were compared with those of the different Leishmania species generated by Fraga et al. . ITS types were assigned to each isolate according to the sequence polymorphism of the 12 microsatellite regions included in ITS1 (four sites) and ITS2 (eight sites), as described by Kuhls et al. . k26 genotypes were assigned according to the size and sequence of the PCR product, following the criteria previously described by Haralambous et al. .
For the generation of unique patterns that could be used for strain identification, we amplified a region of the kinetoplast DNA minicircles and evaluated the restriction profile after Hae III (Roche Applied Science) endonuclease digestion  using DNA extracted from axenic promastigotes and from experimentally infected murine tissues.
Novy-MacNeal-Nicolle medium (NNN) was prepared with a semi-solid phase made of 1.4% agar (Sigma-Aldrich), 0.6% NaCl (Merck), 31% defibrinated rabbit blood, 625 units/mL penicillin, 625 units/mL streptomycin and RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin and 20 mM HEPES buffer (all from Lonza) as liquid phase.
Growth curves and viability
Parasites were first passed in mice to control their virulence and frozen in vials for future use until 10 in vitro passages . Promastigotes were cultivated at 26°C with an initial inoculum of 106 parasites/mL in NNN from a synchronized culture in the same media and followed for 6 days. In each day, parasites were counted in a hemocytometer and stained with Annexin V and 7-amino-actinomycin D (7-AAD) for viability analysis as described in . 10 000 gated events were analyzed in a FACSCanto II (BD Biosciences) and the percentage of Annexin-/7AAD- cells determined with FlowJo software (TreeStar).
In each day of culture, promastigotes were recovered and washed in PBS/FBS 2%. 2×106 parasites were resuspended in 1 mL of PBS/FBS 2% and 3 mL of ice-cold absolute ethanol (Panreac) was carefully added while vortexing. After fixation for 1 hour at 4°C, the parasites were washed in PBS and resuspended in 1 mL of propidium iodide (PI) staining solution consisting of citrate buffer 3.8 mM in PBS, 50 μg/mL PI (Sigma-Aldrich) and 0.5 μg/μL RNAse A (Sigma-Aldrich). Following an incubation of 30 minutes at 4°C, 20 000 single live cells were acquired in a FACSCanto II and analyzed with the FlowJo’s cell cycle built-in tool.
Quantification of metacyclogenesis-dependent gene transcription
107 promastigotes from day 1 to 6 of culture were resuspended in TRIzol reagent (Invitrogen) and frozen at −80°C. Total RNA was extracted using chloroform and isopropanol according to the manufacturer’s instructions and solubilized in 10 μL of nuclease-free water. RNA of high quality was obtained (RQI between 9.0 and 10.0) as assessed using RNA StdSens Chips of the Experion automated electrophoresis system (Bio-Rad). RNA concentration was determined using a Nanodrop ND-1000 spectrophotometer. Samples were stored at −80°C until cDNA was synthesised. Reverse transcription was performed with iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions over 500 ng of total RNA. Meta-1, Small Hydrophilic Endoplasmic Reticulum-associated Protein (SHERP) and histone H4 transcription was quantitatively analyzed after normalization with rRNA45 transcription by qPCR using the iQ SYBR Green Supermix according to the manufacturer’s instructions in a My iCycler iQ5 (Bio-Rad). 4 μL of cDNA (diluted 25×) was used as template that was run in duplicate with 500 nM (Meta-1 and histone H4) or 250 nM (SHERP and rRNA45) of the following primers (from Stabvida): Meta-1 [GenBank: NC_009401] forward: 5′-GGGCAGCGACGACCTGAT-3′ and reverse: 5′-CGTCAACTTGCCGCCGTC-3′ (modified from ); histone H4 [LinJ35.1400, GenBank: XM_001468907] forward: 5′-ACACCGAGTATGCG-3′ and reverse: 5′-TAGCCGTAGAGGATG-3′ ; SHERP [GenBank: XM_003392466] forward: 5′-CAATGCGCACAACAAGATCCAG-3′ and reverse: 5′-TACGAGCCGCCGCTTATCTTGTC-3′ ; rRNA45 [GenBank: CC144545] forward: 5′-CCTACCATGCCGTGTCCTTCTA-3′ and reverse: 5′-AACGACCCCTGCAGCAATAC-3′ . Changes in relative gene expression were determined with ΔΔCT method and results show fold changes comparative to day 1 calculated by 2-ΔΔCT.
In vitro infections
Bone marrow-derived macrophages (BMMo) were produced as described previously . Stationary promastigotes cultivated in NNN for 4 days were washed and put in contact with the cells in 1:10 ratio (cell:parasites) for 4 hours. Extracellular parasites were washed away with PBS and the cells incubated for more 24, 48, 72 or 96 hours or fixed immediately with 2% PFA. The macrophages were mounted on Vectashield with DAPI (Vector Laboratories) and 100 infected cells or 400 total cells were counted in duplicate by fluorescence microscopy in a Zeiss Axioskop (Carl Zeiss). The percentage of infected cells and the geometric mean of the number of parasites per infected cell were evaluated. The infection index was calculated by multiplication of both parameters to account for the overall parasite load.
In vivo infections
7–8 week-old BALB/c male mice (4–5 animals per group, except in 2-week infections with E390M strain where only 3 animals were used) were infected via the intraperitoneal route with 108 promastigotes of each strain cultivated in NNN for four days. After 2 or 6 weeks of infection mice were anesthetized with isoflurane and sacrificed by cervical dislocation. Blood, inguinal lymph nodes, spleen, liver and femoral bone marrow were recovered for quantification of parasite load. Blood and spleen were also used for the evaluation of humoral and cellular responses.
Parasite load quantification
Parasite load was quantified in samples that were collected and frozen at the time of animal sacrifice. We used 200 μL of blood collected with EDTA, 10 mg of spleen and liver (single cell suspensions), 3 × 106 bone marrow cells and the inguinal draining lymph node to extract DNA. First, 400 μL of a buffer containing 10 mM NaCl, 10 mM EDTA and 10 mM Tris–HCl with pH 8.0 were added to the samples, which were incubated overnight with 40 μg of proteinase K (Sigma-Aldrich) at 56°C with shaking. Then, the samples were vortexed and incubated for 20 minutes at 70°C. DNA was extracted using phenol/chloroform/isoamyl alcohol (all from Merck Millipore). After precipitation with ice-cold 70% ethanol solution, DNA was dissolved in 100–200 μL of nuclease-free water. We quantified the total DNA in a Nanodrop ND-1000 spectrophotometer and prepared dilutions of concentrations adjusted for each tissue. We quantified Leishmania sp. DNA by qPCR using 1000 nM of R223 and 500 nM of R333 primers (Sigma-Aldrich) for the small subunit rRNA (SSUrRNA) . Depending on the tissue, 100 to 400 ng of total DNA served as a template in a 20 μL reaction using LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science) according to the manufacturer’s instructions, in a touchdown qPCR performed in a LightCycler 2.0 carousel-based instrument (Roche Applied Science) with final annealing temperature of 65°C . CTs were extrapolated in a standard curve constructed with serial dilutions of L. infantum DNA (strain JPC, MCAN/ES/98/LLM-722) diluted in host DNA (from spleen of naïve mice) to calculate Leishmania content in parasites/μg DNA. Whenever the qPCR gave a positive (with the expected melting curve) but unquantifiable value or a doubtable specific product (aberrant melting curve), we performed a nested PCR  that has a higher sensitivity (0.01 parasites) than the qPCR (0.6 parasites) to confirm the positivity of the quantitative result. 300 nM of R221 and R332 primers  were used for the first amplification reaction. For the second reaction, 10 μL of the first PCR product diluted 1:40 served as template with the same R223 and R333 primers (300 nM and 150 nM, respectively) used for the qPCR. This molecular quantification was applied after proper validation by comparison with limiting dilution assay (Additional file 1: Additional Methods and Additional file 2: Figure S1).
Splenic cell populations
5 × 105 splenocytes were surface-stained for 20 minutes at 4°C with saturating concentrations of monoclonal antibodies (all from Biolegend). After washing twice with PBS/FBS 2%, the cells were examined by flow cytometry in a FACSCanto (BD Bioscences) and analyzed with FlowJo software. After acquisition of 50000 cells identified by FSC and SSC parameters, major populations were identified as follows: CD4+ T lymphocytes (PerCp.Cy5.5 anti-CD3, clone 17A2; APC.Cy7 anti-CD4, clone GK 1.5), CD8+ T lymphocytes (PerCp.Cy5.5 anti-CD3; FITC anti-CD8, clone 53–6.7), B cells (FITC anti-CD19, clone 6D5), monocytes/macrophages (PE.Cy7 anti-CD11b, clone M1/70; PerCp.Cy5.5 anti-Ly6C, clone HK1.4).
The specific humoral response was analyzed by ELISA as described elsewhere . In short, 96-well microtitration plates (Greiner Bio-One) were coated with 10 μg/mL of soluble Leishmania antigens (SLA) in carbonates buffer pH 8.5 and then blocked with PBS/gelatin 1%. Sera were diluted 1:100 and incubated for 2 hours at 37°C. After washing with PBS/tween 20 0.1%, HRP-conjugated anti-IgG1 or anti-IgG2a (Southern Biotech) were added to the wells at a dilution of 1:5000 and incubated for 30 minutes at 37°C. The plates were revealed with 0.5 mg/mL of o-phenylenediamine dihydrochloride (Sigma-Aldrich) in citrate buffer pH 4.0 and the reaction was stopped with HCl 3 N. The absorbance was read at 492 nm in a Synergy 2 microplate reader (Biotek).
Animals and ethics statement
For the in vitro experiments we used 10–12 week-old female BALB/c mice bred and maintained at IBMC - Instituto de Biologia Molecular e Celular (Portugal) animal facilities. For the in vivo experiments 7–8 week-old male BALB/c mice were bred and maintained at the Instituto de Salud Carlos III (Spain) animal facilities. Mice were housed in IVC cabinets with sterile food and water ad libitum. All experiments conducted were carried out in accordance with the IBMC.INEB and ISCIII Animal Ethics Committees and the Portuguese and Spanish National Authorities for Animal Health guidelines that follow the statements on the directive 2010/63/EU of the European Parliament and of the Council. ACS has an accreditation for animal research given from Portuguese Veterinary Direction (Ministerial Directive 1005/92).
GraphPad Prism 5 (GraphPad Software) was used to perform all the statistical analysis. The results are presented as means ± standard deviations (SD). To compare statistical differences between means two-sided t test or one-way ANOVA followed by Dunnett’s multiple comparison test were run when comparing 2 or more groups, respectively, unless otherwise stated. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Molecular characterization of the clinical isolates of L. infantum
To understand the intraspecies polymorphisms and its possible impact on both in vitro and in vivo infectivity, we characterized certain molecular aspects of these four L. infantum strains.
PCR-RFLP of kinetoplast DNA minicircles was used as a tool for creating an individual identity for each strain. Because we were working with L. infantum strains that preferably could have similar growth and morphology, they would be indistinguishable in in vitro cultures. In case of cross-contaminations between strains [29, 30], we would like to have a tool for identification of our parasites. All the strains showed very complex profiles (Figure 1B), but each one of the specimens was clearly identifiable by the examination of the most intense bands.
Characterization of biological features of the promastigotes generated in vitro
In vitro cultivation has a major impact on the virulence of the pathogens due to intrinsic properties of the culture media that modulate Leishmania infectivity (Santarém, N. and Cunha, J., submitted results and [8, 31]), or to the loss of adaptive capacities to mammalian host cells resulting from long-term in vitro cultivation of promastigotes. A relevant experimental bias can be introduced if these factors are not considered. Hence, the comparison of the infective capacity of distinct strains should take into account the adaptation to culture conditions and/or the axenic growth behavior [7, 32].
Evaluation of the infectivity of L. infantum isolates
Estimated overall parasite load of L. infantum -infected BALB/c mice 2 and 6 weeks post-infection
Total parasite load
2 weeks post-infection
6 weeks post-infection
67.7 × 104
8.82 × 104
44.1 × 104
3.81 × 104
62.7 × 104
0.40 × 104
329 × 104
18.9 × 104
157 × 106
2.77 × 106
112 × 106
0.14 × 106
0.35 × 106
2.54 × 106
86.8 × 106
2.42 × 106
15.0 × 107
4.49 × 107
11.9 × 107
50.9 × 107
13.9 × 107
2.29 × 107
18.1 × 107
11.7 × 107
4.83 × 104
0.19 × 104
67.0 × 104
0.10 × 104
1.78 × 104
0.015 × 104
10.3 × 104
0.59 × 104
0.97 × 103
0.42 × 103
19.4 × 103
0.32 × 103
6.54 × 103
3.12 × 103
1.95 × 103
4.53 × 103
3.08 × 108
0.48 × 108
2.32 × 108
5.09 × 108
1.40 × 108
0.25 × 108
2.71 × 108
1.19 × 108
In the chronic phase, 6 weeks after infection, the four strains showed relative infection profiles in the spleen (Figure 5A), bone marrow (Figure 5C) and lymph nodes (Figure 5D) similar to the acute phase. As before, HL was shown to be the most infective strain in spleen and lymph nodes. In the bone marrow the differences were once more not as accentuated between strains, although the higher parasite loads were found in HL infected mice. In the liver (Figure 5B), HL was still the most infective strain, but Bibiano, which in the other tissues presented comparable parasite loads, was, significantly, the least infective strain, whereas E390M and ST produced intermediate infections. In the blood (Figure 5E), the differences observed at 2 weeks post-infection were neutralized, as the four strains showed similar levels of circulating parasites.
Distribution and compartmentalization throughout the infection
Evaluating the progression of the disease, these four strains depicted very different trends. In the acute phase of infection, Bibiano and HL were found in very high numbers in the visceral organs but evolved in the opposite directions with chronicity. Bibiano was efficiently cleared from the liver, with a 425-fold reduction, though in the spleen the parasite load did not alter. As to HL, the liver infection was maintained and the splenic parasite burden increased 5 times from 2 to 6 weeks after infection, which showed not only high capacity to infect but also to perpetuate in the host. E390M, on the contrary, showed a low infective phenotype, with the lowest parasite loads in all the tissues quantified in the acute phase of infection. Through time, this strain was not able to proliferate in the spleen or in the bone marrow; the parasites resisted in the liver and showed a 7.4-fold increase in the blood. Despite with disparate initial parasite loads (more than 6-fold difference), Bibiano and E390M, both isolated from HIV+ patients, followed a very similar trajectory in the progression of the disease and, eventually, would be eliminated over time in these immunocompetent BALB/c mice. The high parasitemia presented by these two strains in the chronic phase may facilitate the anthroponotic transmission of Leishmania between the intravenous drug users (IVDUs) , one of the populations with highest risk of HIV/Leishmania coinfection . Concerning ST, the standard virulent strain in our laboratory, showed a clear tropism for the bone marrow in the acute phase, with the lowest parasite loads in the remaining organs compared to other strains. However, 6 weeks post-infection, ST dramatically multiplied reaching levels ≈ 6- and ≈ 17-fold higher in the spleen and liver, respectively.
As a final remark, this study allowed us to verify that bone marrow parasite load is maintained in a range that does not suffer major alterations either over-time nor is it strain-dependent. Possibly this is the reason why bone marrow aspirates are the eligible sample for leishmaniasis diagnosis, either for microscopic analysis, culture or molecular techniques .
Infectivity relates to cell modulation
The molecular typing strategy confirmed the previous zymodeme characterization by MLEE and provided further knowledge that can be applied to diagnostics and population genetics studies. In this sense, the k26 sequence for E390M strain generated in this work adds information for a zymodeme (MON-284) not included in the study by Harambolous , thus contributing to k26 gene-based typing methodology, which is being used increasingly in population genetics and molecular epidemiology studies related to the L. donovani complex [58, 59]. Laboratory conditions for in vitro culture were set to produce Bibiano, E390M, HL and ST fit promastigotes in the same developmental stage. In vivo infections with HL confirmed the in vitro phenotype of the most infective strain as more parasites were estimated to be present in the whole animal. ST was also considered to be highly infective though with a slower progression over time. Bibiano and E390M, isolated from HIV+ patients, showed differential infectivity and immunomodulation that could be influenced by the initial compartmentalization in host tissues. Interestingly, the most (HL) and the least (E390M) infective strains were the most immunogenic, revealing high levels of anti-Leishmania IgG2a and IgG1, especially in the chronic phase of infection.
This work is in line with previous studies [6, 7, 17, 60] that show that leishmaniasis is a multifactorial disease and the broad spectrum of clinical manifestations depends on the genetics and inherent characteristics of the parasite coordinated with the susceptibility of the host.
We thank Doctor Maria da Luz Duarte from São Marcos Hospital, Braga, Portugal, for kindly providing us with the skin sample infected with HL strain of L. infantum and Joana Tavares from the Parasite Disease Group, IBMC, Porto, Portugal, for the its isolation and preparation of stocks. We thank Carmen Chicharro from WHO Collaborating Center for Leishmaniasis, National Center of Microbiology, Institute of Health Carlos III, Spain, for analyzing its zymodeme. We thank Ricardo Silvestre and Mariana Resende from the Parasite Disease Group, IBMC, Porto, Portugal, for the help in animal experiments and flow cytometry analysis.
This work was funded by FEDER funds through the Operational Competitiveness Programme – COMPETE and by National Funds through FCT – Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-019648 (PTDC/BIA-MIC/118644/2010) and FCOMP-01-0124-FEDER-011054 (PTDC/SAU-FCF/100749/2008) and also MICINN project number PIM2010-ENI00627. JC was supported by fellowship from FCT code SFRH/BD/48626/2008 and CS by Contratos de Técnicos de apoyo a la investigación en el SNS code AES-FIS-2011.
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