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

Abstract

Leishmaniasis is transmitted between mammalian hosts by the bites of bloodsucking vector sand flies. The dose of parasites transmitted to the mammalian host has never been directly determined. We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by individual infected flies (Phlebotomus duboscqi). The number of parasites transmitted varied over a wide range in the 58 ears in which Leishmania were detected and demonstrated a clear bimodal distribution. Most of the infected mice were inoculated with a low dose of <600 parasites. One in four received a higher dose of >1,000 and up to 100,000 cells. High-dose transmission was associated with a heavy midgut infection of >30,000 parasites, incomplete blood feeding, and transmission of a high percentage of the parasite load in the fly. To test the impact of inoculum size on infection outcome, we compared representative high-(5,000) and low-(100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly-derived metacyclic forms of L. major and preexposed the injection site to the bites of uninfected flies. Large lesions developed rapidly in the ears of mice receiving the high-dose inoculum. The low dose resulted in only minor pathology but a higher parasite titer in the chronic phase, and it established the host as an efficient long-term reservoir of infection back to vector sand flies.leishmaniasis ͉ vectors ͉ bites ͉ parasites

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).…”

“…The L. major NIH Friedlin V1 (FV1) strain was originally obtained from the Jordan Valley (MHOM/IL/80/FN). A stable transfected line of L. major FV1 promastigotes expressing a red fluorescent protein (L. major-RFP) was generated as described previously (36). Parasites were grown in vitro at 26°C in medium 199 supplemented with 20% heat-inactivated fetal calf serum (FCS; Gemini Bio-Products), 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml biotin.…”

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

“…Parasite: Although the dose of Leishmania promastigotes delivered by the sand fly vector to the skin of mammalian host has never been directly determined, using a real time-PCR approach it has been possible to reveal a remarkable range of 10 to 100,000 in the dose of parasites transmitted to mice exposed to a single infected sand fly 15 . More recently, another study determined infection parameters in the sand fly vector that predict transmission of L. (L.) major: it was estimated in the range of 10 3 to 10 6 total parasites per fly 34 .…”

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

“…The existence of two distinct patient profi les (classic and severe) might be a consequence of several factors such as the infective strength level 11 , host immunosuppression 8,12,13 , and the genetic background of the host 14,15 , or it might be genetically determined by parasitic virulence factors 16 . The identifi cation of genetic changes in the pathogenic target genes is a starting point in the search for virulence factors 17 .…”