Abstract
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.
Highlights
Protozoan parasites of the genus Leishmania undergo several developmental transitions during their life cycle
The authors demonstrate a correlation between the maintenance of parasite culture with a growing defect of the promastigote form to differentiate in the mammalian amastigote form
This research provides a biological explanation for the loss of virulence due to sustained parasite culture and discusses the impact for all experimental work done with visceral Leishmania species
Summary
Protozoan parasites of the genus Leishmania undergo several developmental transitions during their life cycle. Ingestion of infected macrophages during a blood meal by the sandfly vector leads to the release of intracellular amastigotes into the vector’s midgut. This abrupt change in environment induces the transformation into extracellular procyclic promastigotes. Long-term in vitro growth of drug-resistant parasites was suggested to mediate a loss of the resistance phenotype [5]. This can be due to either loss of virulence factors induced by the lack of a survival pressure or due to disadvantageous adaptations to the media resulting in phenomena similar to clonal selection [6]. One must carefully consider the influence of the different laboratorial factors in order to minimize these variables
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