Visualization of Leishmania tropica Infection in BALB/c Mice by Bioluminescence Imaging

Abstract

Background: Leishmania tropica is the cause of more than one form of leishmaniasis and lacks a known reservoir animal. This study compares the potential infectivity of recombinant and wild-type L. tropica in BALB/c mice.

Methods: The potential infectivity of recombinant L. tropicaEGFP or L. tropicaEGFP-LUC by two different, the subcutaneous and intradermal, routes was compared using a range of classical detection methods and bioluminescence imaging (BLI).

Results: In addition to the results obtained from classical diagnostic approaches, the BLI signals were detected in footpads and ears of L. tropica-infected animals. The BLI revealed that a bioluminescence signal can be observed at the inoculation site. The stability of the BLI remained constant in the footpad, but the signal was detectable for only three months in the pinna due to the decline in infection over time.

Conclusion: The presented data are a precise verification of the assumption that BALB/c mice could be used as an experimental model for L. tropica infectivity.

Fig. 1

Recombinant parasite confirmation. (A) Light microscopy images, (B) fluorescent microscope images, and (C) Western blot analysis. Expression of EGFP-LUC protein (~89 kDa) was confirmed in L. tropica^EGFP-LUC parasite using both antibodies (anti-GFP and anti-LUC), and expression of EGFP protein (~27 kDa) was also confirmed in L. tropica^EGFP through anti-GFP

Fig. 2

Monitoring the spread of the disease and the measurement of parasite burden in the footpad and ear injected with different lines of L. tropica (L. tropica^WT, L. tropica^EGFP, and L. tropica^EGFP-LUC). (A) Measuring the infected footpad’s thickness by a caliper in groups G1, G2, and G3; (B) measuring the diameter of inflammation in the infected ear by a ruler in G4, G5, and G6; (C) parasite load in the popliteal dLNs in G1, G2, and G3; (D) parasite burden in the retromaxillar dLNs in G4, G5, and G6 assessed through the limiting dilution method at four time points (1, 2, 3, and 6 months after the start of the infection). The bars indicate the SD of the mice or dLNs (4-5 individual mice in each group). ^*p < 0.05

Fig. 3

Progressive monitoring of the footpad through BLI in one mouse infected with L. major^EGFP-LUC (left) as a positive control and two (at week 6^th) or three mice infected with L. tropica^EGFP-LUC (right) at different time points, i.e. 6, 7, 12, 17, and 24 weeks after parasite inoculation in the footpad. The first bioluminescence signal-expressing parasite (L. tropica^EGFP-LUC) infection was observed six weeks post-infection, and infection increased with every passing week. The highest signal or rainbow image was apparent 24 weeks after infection. Red and blue areas represent the most and the least intense signals, respectively. In the bottom, the BLI of L. tropica^WT-infected BALB/c mice at 17^th week, as a negative control, has been shown

Fig. 4

The follow-up of parasite infection in the infected ear through BLI in one mouse infected with L. major^EGFP-LUC (left) and three mice infected with L. tropica^EGFP-LUC (right) at 3, 6, 7, and 17 weeks after parasite inoculation. The first bioluminescence signal was observed three weeks after the injection of L. major^EGFP-LUC that was used as a positive control and seven weeks after the inoculation of L. tropica^EGFP-LUC in the ear. The highest intensity in the infected mice by recombinant L. tropica was detected at the seventh week. Next time (at the 17^th week), the intensity of bioluminescence decreased. Regarding the mice infected with recombinant L. major^EGFP-LUC, the first sign of infection was detected at week three, and the strongest signal of BLI was observed 17 weeks after the injection of the parasite. At the 17^th week, infectivity level was detected even in the LNs. Red and blue areas represent the most and least intense signals, respectively. In the bottom, the BLI of L. tropica^WT-infected BALB/c mice at 17^th week, as a negative control, has been shown

Fig. 5.

The quantification of LUC expression in the dLNs of BALB/c mice infected with L. tropica^EGFP-LUC in two different conditions, in vitro and in vivo. (A) Mice were infected with recombinant L. tropica^EGFP-LUC, and the dLNs were isolated from four mice and were homogenized individually in lysis solution 8 and 12 weeks after the injection of the parasite in the footpad. After 5 min, D-luciferin was added to each well as a substrate. Enzymatic reaction was quantified by 1 s/well and a sensitivity of 100 immediately thereafter; (B) the regions of interest from the footpad of the mice infected with L. tropica^EGFP-LUC were quantified in pixels and calculated by the sum of intensity (mean ± SD) at different time points. Data are shown as the mean ± SD of the LUC activity of each dilution in duplicate