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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.

New insights into experimental visceral leishmaniasis: Real-time in vivo imaging of Leishmania donovani virulence