<em>In vivo</em> Imaging of Transgenic <em>Leishmania</em> Parasites in a Live Host

Abstract

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

“…Of these, the luciferase-based systems have proven to be sensitive, rapid, reproducible, and versatile, with tagged parasites now being used to follow the fate of the pathogen during the course of an animal model infection (49)(50)(51)(52). Here, we constructed a luciferase-expressing L. major line making use of an existing T. cruzi integration vector, modifying the expression construct such that it would integrate into the leishmanial ribosomal array (39).…”

Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the ProtozoanLeishmania infantum chagasi

“…Assessment of parasite load by in vivo imaging. Relative burdens of luciferaseexpressing L. infantum chagasi parasites at the inoculation site were determined as previously described (43). Briefly, 1 day after inoculation of neutrophil-depleting or control antibody, metacyclic L. infantum chagasi promastigotes expressing luciferase were introduced into female BALB/c mice intradermally in the ear.…”

“…The total numbers of parasites in tissues were quantified by in vivo imaging (Xenogen IVIS 200 system) (Fig. 6A) (43). The numbers of parasites in the draining lymph nodes were quantified by qPCR (Fig.…”

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

“…The genetic modification of organisms, such as viruses 1,2 , bacteria 3 , or small mammals 4 to either induce or constitutively express bioluminescence, has been highly successful and widely demonstrated [5][6][7] . Bioluminescence, an in vivo chemiluminescent reaction involving naturally occurring reagents, has the advantage of producing light without the need for an external light source.…”