The use of sand flies to initiate experimental infection with Leishmania
has revealed several important biological differences in the host response to infection and disease outcome as compared with needle inoculation 
. However, the number of reports employing experimental transmission of Leishmania
by bite, including evidence of viable transmitted organisms, is scant. This is due, at least in part, to the unpredictability of parasite transmission by sand fly bite, compounded by a shortage of information on the parameters of sand fly infection that influence transmission. In the studies reported here we identified a parameter of Leishmania
infection within the sand fly, the percentage of metacyclic promastigotes, which best correlates with the subsequent frequency of parasite transmission to the mammalian host in an experimental setting. Meta-analysis of our extensive experience involving transmission attempts by 4–5 L. major
-infected flies exposed to the mouse ear dermis permitted us to establish a threshold of metacyclic percentage in infected flies (71.7%) that reliably predicts an acceptable level of transmission success, arbitrarily defined as ≥70%.
The predictive value of the infection parameters reported here should help make experiments relying on sand fly challenge more manageable. In particular, it should prevent the use of populations of infected flies that are unlikely to transmit to a sufficient number of animals to interpret subsequent experimental results. This is of critical importance in experiments where the animals used for challenge, e.g. vaccinated mice, monkeys, or dogs, are valuable and limited.
While increasing the number of flies per animal could be used to compensate for flies harboring sub-optimal numbers of metacyclics, we would argue that over-exposure of the inoculation site to sand fly bites begins to substitute one form of non-physiologic exposure, i.e. needle challenge, for another. This raises the question as to whether the numbers of parasites per midgut and frequencies of metacylics achieved in our experimental system reflect what is found in naturally infected flies. Published data on the intensity and composition of Leishmania
infections within wild-caught sand flies are rare. One study revealed parasite loads of between 101
parasites per fly that appeared to vary according to oviposition status 
, although this was no doubt also influenced by the stage of infection in the fly. Groups of laboratory-reared P. duboscqi
flies with mature experimental L. major
infections average between 102
parasites per midgut depending upon the dose of parasites used for infection 
. Similar results have been reported following experimental infection of Lutzomyia longipalpus
with L. infantum
(Lutz and Neiva) 
and Lu. Longipalpus
with L. mexicana 
. As far as we are aware, there is no published data on the frequency of metacyclic promastigotes within infected wild caught flies. Therefore, we can only speculate that flies infected with L.m.
RYN, which contain in the range of 103
total parasites per fly and high frequencies of metacyclic promastigotes, approximate the infection status of transmitting flies in the wild.
In nature, sand flies likely become infected with varying doses of parasites depending upon their feeding behavior and the concentration of parasites in the lesion or blood upon which they feed. We observed that groups of flies infected with larger initial doses of parasites had heavier infections, were more likely to transmit parasites during a second feeding attempt, and caused more severe disease when transmission occurred. This increased disease severity was likely due to a larger inoculum transmitted by the more heavily infected flies. In the single fly transmissions analyzed by Kimblin et al. 
, there was a direct correlation between the intensity of midgut infections and transmitted dose.
These results also suggest that the number of organisms picked up from an infection reservoir may have direct bearing on the severity of disease resulting from the bite of that infected fly. Thus, reservoir hosts, including humans for the anthroponotic forms of Leishmaniasis, with active infections containing large numbers of parasites may be more likely to give rise to heavily infected flies that will transmit more severe infections. The influence of transmitted dose on infection outcome has been difficult to study because it is impossible to control the number of parasites an individual sand fly deposits in the skin. Our results suggest that groups of flies infected with varying doses of parasites will deposit corresponding doses of parasites upon exposure to the dermis, leading to more or less severe disease.
Finally, we demonstrated a role for infected sand fly oviposition status and environmental conditions at the time of transmission in subsequent transmission frequency. These observations add to those of Rogers et al. 
in which maturation of L. mexicana
infections within Lu. longipalpus
sand flies resulted in more persistent sand fly feeding behavior and enhanced transmission. Together, these experiences should facilitate studies of the host response to Leishmania infection under conditions of experimental sand fly challenge, already severely limited by the few laboratories that have sand flies available for study.