Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.

Chronic Trypanosoma cruzi infections lead to cardiomyopathy in 20-30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue-penetrating orange-red light. This enabled long-term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End-point ex vivo bioluminescence imaging allowed tissue-specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro-intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR-inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long-term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium-specific infection

In vivo imaging of trypanosome-brain interactions and development of a rapid screening test for drugs against CNS stage trypanosomiasis.

HUMAN AFRICAN TRYPANOSOMIASIS (HAT) MANIFESTS IN TWO STAGES OF DISEASE: firstly, haemolymphatic, and secondly, an encephalitic phase involving the central nervous system (CNS). New drugs to treat the second-stage disease are urgently needed, yet testing of novel drug candidates is a slow process because the established animal model relies on detecting parasitemia in the blood as late as 180 days after treatment. To expedite compound screening, we have modified the GVR35 strain of Trypanosoma brucei brucei to express luciferase, and have monitored parasite distribution in infected mice following treatment with trypanocidal compounds using serial, non-invasive, bioluminescence imaging. Parasites were detected in the brains of infected mice following treatment with diminazene, a drug which cures stage 1 but not stage 2 disease. Intravital multi-photon microscopy revealed that trypanosomes enter the brain meninges as early as day 5 post-infection but can be killed by diminazene, whereas those that cross the blood-brain barrier and enter the parenchyma by day 21 survived treatment and later caused bloodstream recrudescence. In contrast, all bioluminescent parasites were permanently eliminated by treatment with melarsoprol and DB829, compounds known to cure stage 2 disease. We show that this use of imaging reduces by two thirds the time taken to assess drug efficacy and provides a dual-modal imaging platform for monitoring trypanosome infection in different areas of the brain

<i>In vitro</i> sensitivity of GVR35-LUC2 compared to WT for different trypanocidal compounds.

†<p>Data show means ± SD of at least three independent assays with duplicate wells.</p

Generation of bioluminescent <i>T. brucei</i> GVR35 for detection of trypanosomes <i>in vivo</i>.

<p>(<b>A</b>) Live GVR35 WT and -LUC2 clones were assessed for luciferase activity <i>in vitro</i> after addition of D-luciferin. The <i>in vitro</i> detection limit was determined by imaging a dilution series of GVR35-LUC2 using IVIS. Data show, on a logarithmic scale, average total flux (in photons per second) ± SD of replicate wells containing a specified trypanosome number. The dotted line indicates background bioluminescence for GVR35 WT. (<b>B</b>) Brains of mice infected with WT or GVR35-LUC2 were compared for trypanosome DNA and neuropathology at different times after infection. Real-time qPCR of the trypanosome TbPFR2 gene was performed on brain homogenates. Neuropathology was scored on haematoxylin and eosin stained sections. Graphs show means and 95% confidence interval for each group (<i>n</i> = 11–12 per group). <i>P</i> value (Student's <i>t</i> test) compared to GVR35 WT is indicated *<i>P</i><0.05; **<i>P</i><0.01.</p

Bioluminescence imaging of <i>T.</i>

<p><b>brucei<i> </i></b><b> GVR35-LUC2- infected mice to assess </b><b><i>in vivo</i></b><b> trypanocidal activity.</b><b> </b> Mice were treated with (<b>A</b>) diminazene aceturate ((i) <i>n</i> = 11, (ii) <i>n</i> = 6), (<b>B</b>) melarsoprol (<i>n</i> = 12), (<b>C</b>) DB75 (<i>n</i> = 6) or (<b>D</b>) DB829 (<i>n</i> = 8) from day 21 and imaged on indicated days after infection. D-luciferin (150 mg/kg) was injected intraperitoneally 10 minutes before imaging. Bioluminescence from the heads of mice is shown as total flux in photons per second (p/sec). ND indicates that trypanosomes were not detected in blood samples. For each treatment images of the same two representative mice over the entire period are shown. In Aii brains were harvested from perfused GVR35-LUC2-infected mice at day 21 (untreated) or day 35 (diminazene-treated), soaked in luciferin and imaged <i>ex vivo</i>. Corresponding untreated heads and treated heads and brains are shown. The same two colour scales are used for all treatments for strong (a) and weaker (b) bioluminescent radiance in photons.second⁻¹.centimeter⁻².steradian⁻¹. The colour scale used is indicated in the top right corner of each image.</p

<i>Ex vivo</i> imaging of bioluminescent <i>T.</i>

<p><b>brucei<i> </i></b><b> GVR35 over the course of infection.</b><b> </b> (<b>A</b>) Brains were removed from perfused GVR35-infected animals at indicated days after infection, soaked in D-luciferin and imaged <i>ex vivo</i>. A comparison between brains from perfused and non-perfused animals at day 14 is shown at the bottom. (<b>B</b>) Organs were harvested from perfused GVR35 WT or -LUC-infected mice at day 35, soaked in D-luciferin and imaged <i>ex vivo</i>. Colour scales indicate bioluminescent radiance. In A, different scales are used for weak (a) and stronger (b) bioluminescent radiance in photons.second⁻¹.centimeter⁻².steradian⁻¹.</p

Trypanosomes invade the meninges.

<p>(<b>A</b>) Lister 427-mCherry trypanosomes (red) in a meningeal blood vessel (outlined by dashed blue lines) at day 3 (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002384#pntd.0002384.s008" target="_blank">Video S1</a>). (<b>B</b>) Extravascular GVR35-mCherry trypanosomes (red) day 13. Blood and collagen appear green. A rapidly moving intravascular trypanosome is indicated by the arrow (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002384#pntd.0002384.s009" target="_blank">Video S2</a>). (<b>C</b>) 24 hr after intravenous (i.v.) injection of DB75 meningeal trypanosomes are labeled and motile (arrows), as are host nuclei (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002384#pntd.0002384.s012" target="_blank">Video S5</a>), day 28. Inset shows a different mouse at day 10, 25 min after i.v. injection of DB75, only the nucleus and kinetoplast are labeled (arrow heads, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002384#pntd.0002384.s010" target="_blank">Video S3</a>). (<b>D</b>) At day 25 numerous GVR35-LUC2 trypanosomes (moving dots in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002384#pntd.0002384.s011" target="_blank">Video S4</a>) are present in the meninges. Labeling by i.v. injection of DB75. (<b>E</b>) Numbers of meningeal trypanosomes plotted against days post-infection. Diminazene (40 mg/kg) at day 21 cleared the meninges of trypanosomes until about day 58 (crosses). (<b>F</b>) Treatment with diminazene at day 21 reduced luciferase emission but by day 44 there was strong emission from the head, despite zero blood parasitemia. Scale in photons.second⁻¹.centimeter⁻².steradian⁻¹. (<b>G</b>) Subsequent multi-photon imaging in area circled in (F) showed no motile trypanosomes in the meninges. I.v. injection of DB75 labeled host nuclei. All scale bars 20 µm.</p

Limit of detection <i>in vitro</i>.

<p>(<b>A</b>) Images of two 96-well microtitre plates containing dilutions of <i>T.b. brucei</i> GVR35 clone VSL2 and a non-transformed control (WT). Each plate was imaged using an IVIS Lumina (Perkin Elmer) with 1 minute exposure and medium binning. Both cell lines were serially diluted from 1×10⁶ to 1×10³ (upper plate) and from 1×10³ to 1×10² parasites ml⁻¹ (cell numbers shown above each plate). 100 VSL2 parasites could be clearly visualised. Note that the imaging software automatically adjusts the heat-map scale to account for the intensity of the well containing the highest number of parasites. (<b>B</b>) <i>In vitro</i> linear regression plots generated from both plates. Each point corresponds to bioluminescence represented by the total flux recorded from a single well. In both cases, linear regression analysis shows a very strong positive correlation between bioluminescence and parasite number (R²>0.99). The graphs show readings from duplicate wells. In the upper graph, duplicate values were extremely close and are not individually distinguishable. In the lower graph, duplicates are shown as red squares and blue triangles, and the dotted line indicates the background in blank wells (green triangles), plus two standard deviations.</p

Monitoring the course of infection.

<p>(<b>A</b>) Bioluminescence (total flux) recorded from two BALB/c mice inoculated i.p. with 3×10⁴<i>T.b. brucei</i> GVR 35 (clone VSL2) vs peripheral blood parasitemia recorded over the course of 36 days. Following infection, the bioluminescence signal fluctuated, peaking at 2×10¹¹ photons/sec/cm² on day 18, and remaining above 5×10¹⁰ photons/sec/cm² until drug intervention. After treatment with berenil on day 32 (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002571#s2" target="_blank">Materials and Methods</a>), total flux and peripheral parasitemia fell rapidly. (<b>B</b>) Ventral and dorsal <i>in vivo</i> imaging of both mice (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002571#s2" target="_blank">Materials and Methods</a>) revealed the rapid growth and dissemination of parasites during the course of infection. Dorsal imaging suggests that as early as day 7, parasites can be detected in the head of both mice. After berenil treatment, the bioluminescent signal was rapidly cleared from the periphery and by day 36 was only weakly detectable. However, there was a strong focal signal localised to the head, particularly apparent when viewed from the dorsal perspective. These mice were sacrificed in accordance with animal welfare regulations. (<b>C</b>) Brains were removed from other mice 33 days post-infection and imaged after perfusion (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002571#s2" target="_blank">Materials and Methods</a>). All brains from infected animals showed a clear bioluminescent signal.</p

Limit of detection <i>in vivo</i>.

<p>(<b>A</b>) Six sets of three BALB/c mice were inoculated i.p. with either 20, 100, 500, 5000, or 50000 bloodstream form <i>T.b. brucei</i> GVR35 clone VSL2. An additional set of three mice was inoculated with 30000 non-transformed parasites (WT). All of the IVIS Lumina images were acquired using large binning, 5 minute exposures, 15 minutes after infection and 10 minutes after administration of luciferin (150 mg kg⁻¹). It was possible to visualise as few as 100 parasites in the intra-peritoneal space. (<b>B</b>) A dose response curve generated from the <i>in vivo</i> limit of detection data. Mean abdominal bioluminescence was recorded from each group. Linear regression analysis shows a very strong positive correlation between bioluminescence and parasite inoculum (R²>0.99). Dotted line indicates mean bioluminescence plus two standard deviations, from mice infected with wild type parasites.</p