Xenodiagnosis of Leishmania donovani in BALB/c mice using Phlebotomus orientalis: a new laboratory model

Background: In areas endemic for visceral leishmaniasis (VL), the majority of infected hosts remain asymptomatic but potentially infectious to biting sand flies. Their infectiousness for sand fly vectors is crucial for the transmission of the disease and can be quantified only by xenodiagnosis. However, in the case of human hosts, xenodiagnosis can be problematic for ethical and logistic reasons. The BALB/c mouse model described in this paper was designed to enable xenodiagnostic studies on VL hosts circumventing the need for human volunteers, it permits xenodiagnosis using the same individual host repeatedly, over several months. Methods: BALB/c mice were intradermally inoculated in the ear pinnae with Leishmania donovani, primarily metacyclic stages isolated from the thoracic midguts of experimentally-infected Phlebotomus orientalis females. Naive sand flies were allowed to feed on anaesthetized mice in 1-3-weeks-interval, firstly on the site of inoculation of L. donovani (weeks 2-8 post infection, p.i.), later on the whole body of mice (weeks 9 - 15 p.i.). Infections of sand flies were evaluated microscopically or by PCR analysis. Results: Although infected mice did not show any signs of disease, 19% (N = 876) of the P. orientalis females that fed at the site of inoculation, became infected. The majority of L. donovani-positive females (76%) had heavy infections with their stomodeal valves colonized by attached parasites. Inoculated mouse ears remained infective for sand flies until week 15 p.i. Females feeding on other parts of the body remained negative with exception of two groups feeding on contralateral ears by week 12 p.i. On week 15, however, these two mice returned negative at xenodiagnosis of the contralateral ears. In sacrificed mice, the highest parasite numbers were found in inoculated ears and their draining lymph nodes. Infections were detected also in the spleen, liver, blood and rarely in the contralateral ear. Conclusions: The study showed that BALB/c mice harbored parasites in sufficient numbers to promote heavy infections in P. orientalis and thus comprised a suitable laboratory model for xenodiagnoses of L. donovani. Parasites persisted in the inoculation site and were found transmissible for months to sand flies biting on the same site.