Characterisation of the ex vivo virulence of Leishmania infantum isolates from Phlebotomus perniciosus from an outbreak of human leishmaniosis in Madrid, Spain

Background: 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. Methods: 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. Results: 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 MO and 115 for infected DC; BOS1FL1-infected cells, 300 for infected MO and 247 for infected DC; and POL2FL7-infected cells, 275 for infected MO and 292 for infected DC. Conclusions: 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.