Characterisation of the ex vivo virulence of Leishmania infantum isolates from Phlebotomus perniciosus from an outbreak of human leishmaniosis in Madrid, Spain
© Domínguez-Bernal et al.; licensee BioMed Central Ltd. 2014
Received: 2 August 2014
Accepted: 23 October 2014
Published: 7 November 2014
Since mid 2009, an outbreak of human leishmaniosis in Madrid, Spain, has involved more than 560 clinical cases. Many of the cases occurred in people who live in areas around a newly constructed green park (BosqueSur). This periurban park provides a suitable habitat for sand flies (the vectors of Leishmania infantum). Indeed, studies of blood meals from sand flies captured in the area showed a strong association between the insect vector, hares or rabbits, and humans in the area. Interestingly, up to 70% of cases have been found in immunocompetent patients (aged between 46-60 years). This study was designed to evaluate the ex vivo virulence of the L. infantum isolates from Phlebotomus perniciosus captured in this area of Madrid.
Murine macrophages and dendritic cells were infected ex vivo with L. infantum strain BCN150, isolate BOS1FL1, or isolate POL2FL7. At different times after infection, the infection indices, cytokine production (IL-12p40 and IL-10), NO release and arginase activities were evaluated.
Using an ex vivo model of infection in murine bone marrow-derived cells, we found that infection with isolates BOS1FL1 and POL2FL7 undermined host immune defence mechanisms in multiple ways. The main factors identified were changes in both the balance of iNOS versus arginase activities and the equilibrium between the production of IL-12 and IL-10. Infection with isolates BOS1FL1 and POL2FL7 also resulted in higher infection rates compared to the BCN150 strain. Infection index values at 24 h were as follows: BCN150-infected cells, 110 for infected MØ and 115 for infected DC; BOS1FL1-infected cells, 300 for infected MØ and 247 for infected DC; and POL2FL7-infected cells, 275 for infected MØ and 292 for infected DC.
Our data indicate that L. infantum isolates captured from this endemic area exhibited high virulence in terms of infection index, cytokine production and enzymatic activities involved in the pathogenesis of visceral leishmaniosis. Altogether, these data provide a starting point for the study of the virulence behaviour of parasites (BOS1FL1 and POL2FL7) isolated from P. perniciosus during the outbreak of human leishmaniosis in Madrid, Spain, and their involvement in infecting immunocompetent hosts.
Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniosis (VL) in humans and other mammals (dogs are the main domestic reservoir hosts) in all countries of the Mediterranean basin ,. VL is a potentially fatal disease in dogs. In several areas of Spain where canine leishmaniosis is highly endemic, seroprevalence of up to 34% has been found, and is considered to be a major problem ,. Other animals including rabbits, hares and cats have also been considered as possible alternative reservoirs of VL -. Parasites are transmitted from vertebrate animals to humans by the bite of infected female phlebotomine sandflies. No effective vaccine exists against any form of human leishmaniosis. The spectrum and efficacy of current antileishmanial drugs are also limited . All these aspects have recently been reviewed in detail elsewhere .
Active VL, mainly diagnosed in young children and in immunocompromised adults, results from uncontrolled multiplication of the parasite in phagocytes of the reticuloendothelial system, and infection is lethal if left untreated. Although the incidence of active disease in humans is relatively low (a range of approximately 5-200 VL cases/year reported by each country), cases are increasing and spreading ,.
The environment is undergoing rapid changes because of human actions  that also contribute to the changing landscape of leishmaniosis. The latter includes increasing risk factors and new scenarios associated with the zoonotic VL ,. In Spain, a district of abandoned farmland and tailings areas surrounded by a large urban population in the southwest of Madrid was chosen as a site for a newly constructed periurban green park (BosqueSur). This area has been associated with an outbreak of human leishmaniosis, and more than 560 human cases have been detected from July 2009 to date; notably, 70% of the cases have been found in immunocompetent patients aged between 46-60 years ,.
Current studies suggest that this outbreak was due to human-induced environmental changes (land cover and land use) that created regional combinations of eco-epidemiological conditions; these conditions may influence patterns of sand fly vector (P. perniciosus) distribution and may result in infections of unusual mammalian hosts of L. infantum-. In this context, xenodiagnostic studies performed with wild lagomorphs (hares and rabbits) captured in the green park demonstrate that these animals play some role as unusual reservoirs that transmit L. infantum parasites to P. perniciosus. Moreover, studies of blood meal preferences of P. perniciosus caught in the area revealed that the insect vector prefers to feed on rabbits and hares, but will occasionally feed on other hosts, such as dogs or humans ,,. Altogether, published data support the idea that the peridomestic and sylvatic transmission cycles overlap in the area, and that infected dogs are not essential to maintaining the transmission cycle of L. infantum.
Since this outbreak started, lagomorphs have attracted the interest of researchers in Spain. Chicharro et al. suggested that the outbreak was not caused by a `new' emerging genotype. Molecular typing studies show that the isolates involved in the outbreak belonged to the ITS LOMBARDI subtype of L. infantum, as did those isolated in different parts of Madrid since at least 1992.
However, the question regarding how parasites have been able to infect a high number of immunocompetent patients during the current outbreak remains important and unanswered. The aim of the present study was to evaluate the ex vivo virulence of the L. infantum isolates recovered from P. perniciosus that were captured in the area of human leishmaniosis in Madrid, in comparison with that of another well-characterized strain.
Mice and parasites
Six-week-old female BALB/c mice were purchased from Harlan Interfauna Ibérica (Barcelona, Spain). The animal research described in this manuscript complied with Spanish (Ley 6/2013) and European Union legislation (2010/63/UE). The protocols used were approved by the Animal Care Committee of Complutense University of Madrid.
L. infantum parasites were used in this study. Two isolates named IPER/ES/2012/BOS1FL1 (BOS1FL1) and IPER/ES/2012/POL2FL7 (POL2FL7) were isolated from P. perniciosus captured in the focus of Madrid using CDC light traps. BCN150 is a well characterized strain (M/CAN/ES/96/BCN150 zymodeme MON-1) that was isolated from a dog with active VL . This strain has traditionally been used in our laboratory's experiments related to VL - and by others in canine leishmaniosis studies ,,. All L. infantum parasites were previously passed through golden hamsters (Mesocricetus auratus). Two months after infection, infected spleen samples were cultivated in NNN medium at 26-27°C for 4-7 days, until promastigotes expanded. These were then used for ex vivo infection experiments in this study.
Differentiation of bone marrow-derived cells
Macrophages (BMMØ) and dendritic cells (BMDC) were differentiated in vitro from bone marrow stem cell progenitors. Briefly, BALB/c mice were euthanised, and cell suspensions were obtained by flushing the femurs and tibias with Hank's Balanced Salt Solution (HBSS). Cells were collected by centrifugation and cultured in DMEM containing 2 mM L-glutamine, 0.1 mM nonessential amino acids, 10 mM HEPES, antibiotics, and 10% heat-inactivated foetal bovine serum (FCS). BMMØ were cultured in non-tissue culture treated Petri dishes for 8 days in the presence of M-CSF (Peprotech) at a concentration of 50 ng/ml. At days 3 and 6, half of the supernatants were discarded and replaced with 5 ml of fresh medium containing M-CSF (50 ng/ml). BMDC were cultured for 10 days in 25 ml flasks in the presence of GM-CSF (20 ng/ml, Peprotech). This supplemented DC medium was added at days 0, 3 and 6. After their respective periods of differentiation, BMMØ and BMDC were obtained as previously described , and displayed a phenotype highly enriched in F4/80+ or CD11c+ cells (~95%), respectively.
Infection index of bone marrow-derived cells
BMMØ and BMDC were cultured overnight in the presence or absence of IFN-γ (500 ng/ml) into LabTek culture chamber slides (Thermo Scientific) using 5 × 104 cells per chamber. On the following day, parasites were added to cells at a ratio of 10:1 parasites: BMMØ or BMDC. After 4 h of incubation at 37°C, a time point that reflects initial infection, extracellular parasites were removed by washing, and cells were incubated in fresh medium for 24 h, 48 h and 72 h. After Giemsa staining, cells were mounted with Coverquick, and 400 cells were counted in duplicate in a microscope Olympus BX41. The percentage of infected cells and the mean of the number of amastigotes per infected cell were evaluated. The infection index was calculated by multiplication of both parameters to account for the overall parasite load, as previously described .
Cytokine production, nitric oxide (NO) release and arginase activity
BMMØ and BMDC (1 × 106 cells/ml) were cultured overnight into 24 well non-tissue culture treated plates. Thereafter, parasites were added (at a ratio of 10:1, as above) to cells and extracellular promastigotes were removed by washing after 4 h. Subsequently, cells were stimulated or not with LPS (1 μg/ml). In some cases, culture supernatants were collected after 24 h for cytokine quantification (IL-12p40 and IL-10, BD Pharmigen) by ELISA according to the manufacturer's instructions. At 96 h, supernatants from the other wells were collected and nitric oxide NO release was measured as nitrite concentration using Griess reagent. Then, BMMØ and BMDC were then harvested and used to determine of arginase activity as previously described .
Statistical analyses were performed using SigmaPlot version 11.0 (Systat Software, Inc). Significant differences between different strains were determined and are designated with asterisks as follows: *P <0.05, **P <0.01, ***P <0.001.
Results and discussion
In this study, we defined virulence in terms of measuring the abilities of a well-characterised L. infantum strain and two isolates from P. perniciosus to induce factors contributing to disease under controlled conditions. In this ex vivo context, we quantified virulence, using assessment of both infection index and the quality of immune responses due to infection, as shown below.
Evaluation of infection index and related markers
4 h after infection
252 ± 50
396 ± 50*
552 ± 50*
319 ± 25
399 ± 30*
475 ± 50*
24 h after infection
110 ± 50
300 ± 50*
275 ± 50*
115 ± 25
247 ± 20*
312 ± 30*
72 h after infection
30 ± 10
60 ± 15*
40 ± 10*
102 ± 10
137 ± 15*
292 ± 20*
Characterisation of the quality of immune response due to ex vivo infection
Infection with L. infantum is known to lead to cytokine secretion by host cells that will dictate the nature of T-cell responses. The quality of these immune responses is influenced by the equilibrium between the production of pro-inflammatory cytokines (mainly represented by IL-12) and anti-inflammatory cytokines (mainly represented by IL-10). In VL, IL-10 suppresses IFN-γ and IL-12 production, while disease regression is associated with optimal IL-12p40 production by DC and MØ that drives a predominant T helper 1 response. Th1 T cell responses result in IFN-γ production, type 1 antibody responses, classical macrophage activation and subsequent NO-mediated killing effector functions ,. These activities are essential to destroy the invading pathogens ,.
This study was designed to examine the ex vivo virulence of L. infantum strains isolated from an area associated with human leishmaniosis in Madrid and to assess the potential implications in this human leishmaniosis outbreak. Our data indicate that L. infantum isolates from an endemic area exhibited high virulence in terms of infection index, cytokine production and enzymatic activities involved in the pathogenesis of VL. These data provide a starting point for the study of the virulence behaviour of parasite isolates (BOS1FL1 and POL2FL7) from an outbreak of human leishmaniosis in Madrid (Spain), including their involvement in infecting immunocompetent hosts. Further studies will be needed to determine whether these findings are observed in an in vivo immunocompetent murine model of VL.
GDB help to design he study, participated in the in vitro studies and helped draft the manuscript. MJ and RM obtained parasite isolates from P. perniciosus captured in the area, participated in data collection and analyses, and helped to draft the manuscript. LOG performed image capture using optical microscopy and helped draft the manuscript. AMR and AM helped to carry out ex vivo infections and participated in the microscopic examination of samples. MTC helped to draft the manuscript. JC conceived and coordinated the study, participated in its design and in the in vitro assays, helped to perform the statistical analyses and drafted the manuscript. All authors read and approved the final manuscript.
This study was supported by Grants AGL2010-17394 and AGL2013-44100R from the Spanish Ministry of Economy and Competitiveness and was partially funded by EU grant FP7-2011-261504 EDENext and the paper is catalogued by the EDENext Steering Committee as EDENext 276 (http://www.edenext.eu).
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