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Experimental infections with visceral  Leishmania  spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of  Leishmania  spp.  in vitro  results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the  in vitro  and  in vivo  infection and immunological profile of  L. infantum  stationary promastigotes submitted to successive periods of  in vitro  cultivation. To evaluate the effect of axenic  in vitro  culture in parasite virulence, we submitted  L. infantum  promastigotes to 4, 21 or 31 successive  in vitro  passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of  in vitro  passages. We validated these  in vitro  observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of  in vitro  passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive  in vitro  passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic  in vitro  culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying  Leishmania  species.

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence