In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Summary

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

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.

Protocol

1. Infection of small animals with transgenic Leishmania

1. Parasite lines

Transgenic Leishmania spp. parasites expressing luciferase are generated using an episomal or an integrating vector as reported.^{1 2} Clonal lines are preferred. Two important points are:

(a) Integrated luciferase is preferred over episomal luciferase, since in theory these parasite lines should better retain the transgene in the absence of drug pressure, i.e. after introduction into a mammal. However even integrated transgenes can be lost at low rates,³ so it is critical to m....

Discussion

The in vivo imaging system (IVIS) provides a method for whole animal imaging or in vivo imaging experimental infection models of different forms of leishmaniasis.^18,16 The Leishmania spp. parasites can be engineered to express firefly luciferase at a level that is detected in vivo with the IVIS imaging technology. One of the major advantages of this method is that it allows non-invasive visualization of Leishmania spp. inside the live animal host. This method has be.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
D-Luciferin Potassium Salt	Reagent	Caliper Life Sciences	122796
IVIS Imaging System 200 Series	Equipment	Caliper Life Sciences		Other IVIS models that can be used include: Lumina II, Lumina XR, Kinetic, and Spectrum.