Evolution of Lesion Formation, Parasitic Load, Immune Response, and Reservoir Potential in C57BL/6 Mice following High- and Low-Dose Challenge withLeishmania major

Abstract

A model of cutaneous leishmaniasis using 10 2 Leishmania major metacyclic promastigotes inoculated into the footpads of genetically resistant C57BL/6 mice was studied in order to more accurately reproduce the evolution of lesion formation and the kinetics of parasite growth and immune response as they might occur in naturally exposed reservoirs and in human hosts. In contrast to the more conventional experimental model employing 10 6 metacyclic promastigotes, in which the rapid development of footpad lesions was associated with an increasing number of amastigotes in the site, the low-dose model revealed a remarkably "silent" phase of parasite growth, lasting approximately 6 weeks, during which peak parasitic loads were established in the absence of any overt pathology. Footpad swelling was observed after 6 weeks, coincident with the onset of parasite clearance and with production of high levels of interleukin-12 (IL-12) and gamma interferon (IFN-␥) in draining lymph nodes. Low-dose challenge of IL-12-and IFN-␥-depleted or -deficient mice provided strong evidence that the induction or expression of cellular immunity is essentially absent during the first 6 to 8 weeks of intracellular growth, since the concentration of amastigotes in the site was not enhanced compared to that for wild-type animals during this time. By monitoring the ability of infected mice to transmit parasites to vector sand flies, it was observed that following low-dose challenge, footpads without apparent lesions provided an efficient source of parasites for exposed flies and that the low-dose challenge actually extended the duration of parasite transmissibility during the course of infection.Leishmania major infection in different inbred mouse strains is a widely used model for the study of Th1 or Th2 responses that control resistance or susceptibility, respectively, to this intracellular parasite (15,30). The resistant C57BL/6 mouse, in particular, is believed to be a relevant model of L. major infections in humans, which are characterized by the development of localized cutaneous lesions that spontaneously heal. The model has been extremely successful in defining the cells, cytokines, and effector molecules involved in acquired resistance, which to date has been shown to require the interleukin-12 (IL-12)-driven activation of Th1 cells for production of high levels of gamma interferon (IFN-␥) which in turn activates leishmanicidal mechanisms in infected macrophages (3,12,16,32,33). In the mouse model, which has typically employed high doses (10 5 to 10 7 ) of promastigotes inoculated subcutaneously into the footpad, the onset of lesion formation, high tissue parasite burden, and immunity is relatively rapid compared to that with natural infection. As a consequence, the model has not been as informative in clarifying the relationships between lesion formation, parasite numbers, and immune response.A model of cutaneous leishmaniasis resulting from low-dose challenge of genetically resistant mice would more closely mimic the evolution of self-...

Quantification of the infectious dose ofLeishmania majortransmitted to the skin by single sand flies

“…Assuming, based on prior studies (6,8,20), that the majority of fly-transmitted parasites are metacyclic promastigotes, needle injection of 5,000 sand fly-derived metacyclics produced lesions that were rapid in onset and relatively severe in size and duration but that eventually healed. By contrast, 100 metacyclics produced only slight pathology, but interestingly, and consistent with a prior study (21), the number of parasites persisting in the skin during the chronic phase was higher in the mice receiving the low-dose challenge. We were able to demonstrate formally that that the low-dose infection established the host as a long-term reservoir of infection back to the vector, at least as efficient as the mice receiving the high-dose challenge.…”

Site-Dependent Recruitment of Inflammatory Cells Determines the Effective Dose of Leishmania major

“…Our observations demonstrate that the number of parasites that establishes infection must be considered in interpreting the influence of the site of infection, as suggested previously (6). We and others have demonstrated that the relative differences in parasite load or lesion size following inoculation at different sites or with different doses of parasites can change depending on the time of analysis (1,3,7,36,48). For example, while we observed earlier control of parasite numbers in the ear than in the footpad, likely due to the earlier onset of adaptive immunity as shown here, a 10-fold-higher parasite load was maintained in the ear during chronic infection (3).…”

“…Here we demonstrate that even before different antigen-presenting cells begin to prime adaptive immunity, the site of inoculation immediately influences the effective dose of L. major parasites that establishes infection. The parasite dose has far-reaching implications for Leishmania infections, including the nature of the adaptive immune response, and is likely one of the most important variables in determining the kinetics and outcome of infection (6,36,(44)(45)(46)(47)(48)). Our observations demonstrate that the number of parasites that establishes infection must be considered in interpreting the influence of the site of infection, as suggested previously (6).…”

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused bY Leishmania (Leishmania) mexicana

“…Conflicting data may arise in part because different parasite strains or species are being examined, different tissue target (the mice's footpad, the ear, or base of the tail) are being infected, and different doses ("low" 1x10 2 and "high" 1x10 6 ) of metacyclic promastigotes have been inoculated 20,27,28 . In addition immunological studies carried out in humans did not reproduce the Th1/Th2 dichotomy described in the L. major, C57BL/6 and the BALB/c mouse model of CL 18,23,24 . In summary there is not an experimental model to study LCL caused by L. (L.) mexicana.…”