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Visceral leishmaniasis is an insidious neglected disease with worldwide distribution. It is caused by parasites from the Leishmania donovani complex, which are able to be transmitted by different species of phlebotomine sand flies and to infect numerous mammal hosts. Despite the high number of people infected or at risk, and the remarkable quantity of studies focusing on this disease, a proper experimental model to efficiently decipher the infectious process of visceral leishmaniasis taking into account the nuances of parasite’s virulence and the duration of the infection is still lacking. Therefore, using golden Syrian hamsters and BALB/c mice, state-of-the-art genetic manipulation applied on a fully virulent L. donovani strain and in vivo imaging approaches, we describe herein three benefits for experimental visceral leishmaniasis: (i) the development of a double transfected bioluminescent (firefly luciferase) and fluorescent (E2-crimson) virulent strain of L. donovani (Ld1S_luci_E2-crimson), favoring a wide range of both in vivo and in vitro investigations, (ii) the establishment of a non-invasive mouse model to evaluate the infectious process during visceral leishmaniasis and the parasite’s virulence in real time, allowing longitudinal studies with the same animals, and (iii) the elaboration of a suitable method to reinstate (and verify anew) the virulence in a population of attenuated parasites, by recovering persistent parasites from chronic infected mice. Consequently, these results open up new perspectives on the study of visceral leishmaniasis, especially in the fields of therapeutics and vaccinology, since the model described herein renders now possible long-lasting follow up studies, with easy and accurate day-by-day verifications of the infection status along with a reduced number of laboratory animals., Trial Registration: ClinicalTrials.gov 2013-0047.

Background: After loading with live Leishmania (L) amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism., Methodology/principal Findings: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i) long-chain fatty acids (LCFA) and cholesterol uptake/transport, (ii) LCFA and cholesterol (re)-esterification to triacyl-sn-glycerol (TAG) and cholesteryl esters (CE), respectively. As these neutral lipids are known to make up the lipid body (LB) core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes., Conclusions/significance: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

Background/objectives: The inoculation of a low number (10(4)) of L. amazonensis metacyclic promastigotes into the dermis of C57BL/6 and DBA/2 mouse ear pinna results in distinct outcome as assessed by the parasite load values and ear pinna macroscopic features monitored from days 4 to 22-phase 1 and from days 22 to 80/100-phase 2. While in C57BL/6 mice, the amastigote population size was increasing progressively, in DBA/2 mice, it was rapidly controlled. This latter rapid control did not prevent intracellular amastigotes to persist in the ear pinna and in the ear-draining lymph node/ear-DLN. The objectives of the present analysis was to compare the dendritic leukocytes-dependant immune processes that could account for the distinct outcome during the phase 1, namely, when phagocytic dendritic leucocytes of C57BL/6 and DBA/2 mice have been subverted as live amastigotes-hosting cells., Methodology/principal Findings: Being aware of the very low frequency of the tissues' dendritic leucocytes/DLs, bone marrow-derived C57BL/6 and DBA/2 DLs were first generated and exposed or not to live DsRed2 expressing L. amazonensis amastigotes. Once sorted from the four bone marrow cultures, the DLs were compared by Affymetrix-based transcriptomic analyses and flow cytometry. C57BL/6 and DBA/2 DLs cells hosting live L. amazonensis amastigotes do display distinct transcriptional signatures and markers that could contribute to the distinct features observed in C57BL/6 versus DBA/2 ear pinna and in the ear pinna-DLNs during the first phase post L. amazonensis inoculation., Conclusions/significance: The distinct features captured in vitro from homogenous populations of C57BL/6 and DBA/2 DLs hosting live amastigotes do offer solid resources for further comparing, in vivo, in biologically sound conditions, functions that range from leukocyte mobilization within the ear pinna, the distinct emigration from the ear pinna to the DLN of live amastigotes-hosting DLs, and their unique signalling functions to either naive or primed T lymphocytes.

Background: Because topical therapy is easy and usually painless, it is an attractive first-line option for the treatment of localized cutaneous leishmaniasis (LCL). Promising ointments are in the final stages of development. One main objective was to help optimize the treatment modalities of human LCL with WR279396, a topical formulation of aminoglycosides that was recently proven to be efficient and safe for use in humans., Methodology/principal Findings: C57BL/6 mice were inoculated in the ear with luciferase transgenic L. major and then treated with WR279396. The treatment period spanned lesion onset, and the evolution of clinical signs and bioluminescent parasite loads could be followed for several months without killing the mice. As judged by clinical healing and a 1.5-3 log parasite load decrease in less than 2 weeks, the 94% efficacy of 10 daily applications of WR279396 in mice was very similar to what had been previously observed in clinical trials. When WR279396 was applied with an occlusive dressing, parasitological and clinical efficacy was significantly increased and no rebound of parasite load was observed. In addition, 5 applications under occlusion were more efficient when done every other day for 10 days than daily for 5 days, showing that length of therapy is a more important determinant of treatment efficacy than the total dose topically applied., Conclusions/significance: Occlusion has a significant adjuvant effect on aminoglycoside ointment therapy of experimental cutaneaous leishmaniasis (CL), a concept that might apply to other antileishmanial or antimicrobial ointments. Generated in a laboratory mouse-based model that closely mimics the course of LCL in humans, our results support a schedule based on discontinuous applications for a few weeks rather than several daily applications for a few days.