In vivo imaging of mice infected with bioluminescent Trypanosoma cruzi unveils novel sites of infection
© Henriques et al.; licensee BioMed Central Ltd. 2014
Received: 20 September 2013
Accepted: 21 February 2014
Published: 3 March 2014
The development of techniques that allow the imaging of animals infected with parasites expressing luciferase opens up new possibilities for following the fate of parasites in infected mammals.
D-luciferin potassium salt stock solution was prepared in phosphate-buffered saline (PBS) at 15 mg/ml. To produce bioluminescence, infected and control mice received an intraperitoneal injection of luciferin stock solution (150 mg/kg). All mice were immediately anesthetized with 2% isofluorane, and after 10 minutes were imaged. Ex vivo evaluation of infected tissues and organs was evaluated in a 24-well plate in 150 μg/ml D-luciferin diluted in PBS. Images were captured using the IVIS Lumina image system (Xenogen). Dissected organs were also evaluated by microscopy of hematoxylin-eosin stained sections.
Here we describe the results obtained using a genetically modified Dm28c strain of T. cruzi expressing the firefly luciferase to keep track of infection by bioluminescence imaging. Progression of infection was observed in vivo in BALB/c mice at various intervals after infection with transgenic Dm28c-luc. The bioluminescent signal was immediately observed at the site of T. cruzi inoculation, and one day post infection (dpi) it was disseminated in the peritoneal cavity. A similar pattern in the cavity was observed on 7 dpi, but the bioluminescence was more intense in the terminal region of the large intestine, rectum, and gonads. On 14 and 21 dpi, bioluminescent parasites were also observed in the heart, snout, paws, hind limbs, and forelimbs. From 28 dpi to 180 dpi in chronically infected mice, bioluminescence declined in regions of the body but was concentrated in the gonad region. Ex vivo evaluation of dissected organs and tissues by bioluminescent imaging confirmed the in vivo bioluminescent foci. Histopathological analysis of dissected organs demonstrated parasite nests at the rectum and snout, in muscle fibers of mice infected with Dm28c-WT and with Dm28c-luc, corroborating the bioluminescent imaging.
Bioluminescence imaging is accurate for tracking parasites in vivo, and this methodology is important to gain a better understanding of the infection, tissue inflammation, and parasite biology regarding host cell interaction, proliferation, and parasite clearance to subpatent levels.
Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that still causes 10,000 to 14,000 deaths per year and affects millions of people in Central and South America, where 8 to 10 million people are infected . T. cruzi has a complex life cycle that involves the following three developmental stages: amastigote (intracellular replicative form in vertebrate host), epimastigote (extracellular replicative form found in the gut of invertebrate host), and trypomastigote (infective form for vertebrate hosts) . It has been shown that T. cruzi is able to infect all nucleated mammalian cells in culture. In vivo it has been shown that even adipocytes can be infected by trypomastigotes . However, cells of the reticuloendothelial, nervous and muscle systems appear to be the preferential ones in the experimental model and in T. cruzi-infected individuals [4, 5].
For a more complete understanding of the tissue distribution of T. cruzi in the entire animal and the evolution of the infection during the course of analysis of new anti-parasite drugs, it is important to establish experimental models in which the parasites can be easily followed within the animal. Currently available chemotherapeutic agents for Chagas disease are nifurtimox and benznidazole. Although both are active in the acute phase, their efficacy is very limited during the chronic phase. In addition, both drugs demonstrate poor activity against many T. cruzi isolates, and have considerable side effects that can lead to the discontinuity of the therapy . In recent years, several studies attempted to identify selective metabolic targets for new drugs, making this a very active area of research. Enzymes and metabolic pathways found by proteomic, transcriptomic, and metabolomic analysis [7–9], coupled with platforms of drug design, necessitate more advanced methods of screening compounds in vivo. Novel methodologies and technologies have been developed for in vitro screening of compounds with inhibitory activity against protozoa, such as viability and proliferative assays . This includes the use of transgenic parasites expressing fluorescent proteins and/or luciferase, in conjunction with sensitive imaging systems and instruments.
The bioluminescent imaging represents an innovation in pre-clinical evaluation of drugs in vivo and in the follow-up of infection in small mammals. The foci of infection can be tracked throughout the body in real time, as long as the animal is alive. This noninvasive technology gives light to the new sites of infection that could not be observed easily through histological techniques and allows investigators to evaluate the dynamic of parasite distribution in the whole body, a methodology that does not require killing the animal to evaluate infected tissues. Some studies on standardization of this important technological tool, using Trypanosoma cruzi, Leishmania sp, and Toxoplasma gondii, have shown its application [11–14]. We previously described the use of bioluminescence imaging to follow the fate of T. cruzi in the invertebrate host . In the present work, we explored, using a bioluminescent imaging system, the progression of the infection in mice infected with transgenic Dm28c-luc strain of Trypanosoma cruzi expressing luciferase. It was possible to monitor in real time the progression of infection in the same group of infected mice. This innovative technology enabled a better evaluation of the acute infection, including the discovery of tissues and organs with intense parasitism during the course of experimental infection up to the chronic phase of the disease. Bioluminescent imaging is a powerful tool for the evaluation of the effect of anti-parasite compounds on the evolution of ongoing biological processes in live mammals and other organisms.
We used wild type Dm28c clone(Dm28c-WT) of T. cruzi and genetically modified parasites expressing luciferase (Dm28c-luc) (description below). Epimastigote forms of both strains were cultivated in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum (FCS) at 28°C up to the logarithmic stage of growth . Metacyclic trypomastigote forms were then obtained in vitro by subjecting T. cruzi epimastigotes from the late exponential growth phase (at cell density of 3 × 107 cells/ml) to nutritional stress in triatomine artificial urine (TAU) medium (190 mM NaCl, 8 mM phosphate buffer, 17 mM KCl, 2 mM MgCl2, pH 6.0) for 2 hours. Metacyclic parasites were further cultivated for five days in TAU medium supplemented with amino acids and glucose (TAU3AAG) (TAU supplemented with 0.035% sodium bicarbonate, 10 mM L-proline, 50 mM sodium glutamate, 2 mM sodium L-aspartate, and 10 mM glucose) . Metacyclic parasites were used to infect LLC-MK2 cells and trypomastigote forms released from cell cultures were used to infect mice.
Intracellular amastigotes and amastigotes were obtained from LLC-MK2 cells infected with trypomastigotes and cultivated for 5 to 10 days in RPMI 1640 medium supplemented with 5% FCS at 37°C in a 5% CO2 atmosphere.
Dm28c-luc trypomastigotes and/or amastigotes of cell culture, maintained in culture for 5 months without G418, were differentianted back to epimastigotes after incubation in LIT medium supplemented with 10% FCS at 28°C. Bioluminescence in epimastigotes were evaluated in a microplate reader Spectra Max2.
LLC-MK2 cells were plated on 13 mm round glass coverslips in a 24 well microplate, maintained for 18 hours in RPMI 1640 medium with 5% FBS at 37°C in a 5% CO2 atmosphere. Afterwards, the cells were washed and allowed to interact with trypomastigotes of Dm28c-WT or Dm28c-luc with a parasite-host cell ratio of 10:1, in 200 μl of RPMI at 37°C in a 5% CO2 atmosphere. After 4 hours, cultures were washed to remove non-internalized parasites and maintained in RPMI 1640 medium with 5% FBS at 37°C in a 5% CO2 atmosphere for 24, 48 and 72 hours. Infected cells were fixed with Bouin, washed with 70% ethanol, water and stained with Giemsa. Subsequently, coverslips were dehydrated in acetone–xylol (100% acetone; 70% acetone – 30% xylol; 30% acetone–70% xylol; 100% xylol) and sealed with Entelan® (Merck). The percentage of infection and number of parasites per infected cells were quantified in at least 500 cells in a light microscope (Leica Microsystems). Two independent experiments were performed in triplicate.
Animals and infection
Seven-week-old male BALB/c mice were obtained from the Animal Laboratory Breeding Center at Fundação Oswaldo Cruz (CECAL) and housed for 7 days in the Laboratory of Cellular Ultrastructure at UFRJ under environmental and sanitation conditions established in the guide for the Care and Use of Laboratory Animals (DHEW publication No. [NIH] 80–23). This project was approved by the Biophysics Institution Committee of Ethics in Animal Research (IBCCF106), according to resolution 196/96 of the National Health Council of the Brazilian Ministry of Health. Experimental groups were composed of five mice, which were intraperitoneally infected with 1 × 106 or 1 × 105 Dm28c-WT or Dm28c-luc parasites in 200 μl of RPMI medium. Parasitemia was determined in 5 μl of blood obtained from tail snips, according to the Pizzi-Brenner method .
Expression of firefly luciferase (Fluc) in Trypanosoma cruzi
The luciferase gene was amplified by PCR using specific primers, the forward containing an XbaI site and the kozak sequence upstream from the start codon (italics), 5′-GCTCTAGA GCCACC ATGGAAGACGCCAAAAACATAAAG–3′ (F-luc), and the reverse primer containing an XhoI site (italic) 5′-CCGCTCGAG CGGTTACACGGCGATCTTTCC-3′ (R-luc), using the Tli DNA Polymerase (Promega) and the following PCR conditions: 94°C, 5 min; 94°C, 30 sec; 60°C, 30 sec; 72°C, 2 min, 30 cycles; 72°C, 10 min. The PCR product was cloned into the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), digested from the TOPO vector, and subcloned into the integrative pTREX vector at the XbaI and XhoI restriction sites . The construction was sequenced on an ABI 3730 Genetic Analyzer (Applied Biosystems) using the sequencing platform installed at the Oswaldo Cruz Foundation (Fiocruz).
T. cruzi epimastigote forms of Dm28c clone (Dm28c-WT) were suspended at 1 × 108 cells/mL in electroporation buffer (EPB) containing 137 mM NaCl; 5 mM KCl; 0.7 mM Na2HPO4; 6 mM glucose; 21 mM HEPES, pH 7.3. The cellular suspension (400 μl) was mixed with 50 μg of plasmid, placed in a 0.2 cm cuvette and subjected to a pulse of 0.45 kV, 500 μF at room temperature in a Gene Pulser apparatus (BioRad Laboratories) . The parasites were resuspended in LIT medium and stable transformants were selected with 200 to 500 μg/mL of G418. Thereafter, positive epimastigotes were selected by serial dilution in a 96 plate, and selected by bioluminescence emission with SteadyGlo reagent (Promega) in a SpectraMax2 microplate reader.
In vitro bioluminescent imaging
To perform a bioluminescent curve of intracellular Dm28c-luc amastigotes, LLC-MK2 cells (103) were plated in a 96 well microplate and maintained for 18 hours in RPMI 1640 medium with 5% FBS at 37°C in a 5% CO2 atmosphere. Afterwards, the cells were washed and allowed to interact with 102 to 104 trypomastigotes of Dm28c-luc. Infected cells were cultivated for 24 to 48 hours in RPMI 1640 medium with 5% FBS at 37°C in a 5% CO2 atmosphere. Bioluminescent images were obtained from trypomastigotes, and from intracellular parasites, after 24 and 48 hours of cultivation. Images were acquired after 5 minutes of incubation of trypomastigotes with 150 μg/ml of D-luciferin in 200 μl of RPMI medium in a black 96 well microplate. Intracellular amastigotes were removed from the plate with 50 μl of SteadyGlo reagent (Promega), transferred to the 96 well black microplate and the volume adjusted to 200 μl of RPMI with 150 μg/ml of D-luciferin. Bioluminescence was quantified with automatic measurement ROI.
In vivo bioluminescent imaging
The infection with transgenic parasites was also monitored over time by detecting the bioluminescence in the whole animal using the IVIS Lumina image system (Xenogen). The equipment consists of a cooled charge-coupled camera mounted on a light-tight chamber with a nose cone delivery device to keep the mice anesthetized during image acquisition (IVIS 100; Xenogen). D-luciferin potassium salt (Xenogen) stock solution was prepared in phosphate-buffered saline (PBS) at 15 mg/ml, filter-sterilized, and stored in a-80° freezer. To produce bioluminescence, mice infected with Dm28c-luc and infected with Dm28c-WT received an intraperitoneal injection of D-luciferin stock solution (150 mg/kg). All mice were immediately anesthetized in an oxygen-rich induction chamber with 2% isofluorane, and images were captured after 10 minutes incubation to allow substrate distribution. Mice infected with Dm28c-WT were injected with D-luciferin (150 mg/kg) or PBS and submitted to the same procedure in the induction chamber, but images were acquired just after 1 hour and 24 hours post infection (1 dpi).
Bioluminescent images were obtained after 1 hour, at 1, 4 to 28, and 180 dpi (chronic phase). Bioluminescent images were obtained of mice in dorsal, ventral, and lateral position. Anesthesia was maintained during the entire imaging process by using a nose cone isofluorane-oxygen delivery device in the light-tight chamber.
To acquire the bioluminescent images, some parameters were considered based on the level of bioluminescent emission, such as the time of exposure, which ranged from a few seconds to 5 minutes, the number of pixels, and the field of view (FOV) with automatic focus. The Living Image software automatically co-registered the luminescent images, which were taken in darkness and displayed in pseudo-colors that represent the intensity of the signal and the photographic image, generating an overlay image.
Ex vivo bioluminescent imaging
Mice infected with 1 × 105 Dm28c-luc trypomastigote forms were euthanized at 14, 21, 28, and 180 dpi (6 months), and selected organs were excised. The bioluminescence in infected tissues and organs was evaluated in a 24-well plate or in individual plates in 150 μg/ml D-luciferin diluted in enough volume of PBS, to cover the tissues, 0.5 ml for 24 well plate and 1 to 2 ml in individual plates. Images were captured using the IVIS Lumina image system (Xenogen), after 5 minutes incubation with substrate.
In vivo and ex vivo bioluminescent image quantification
To measure photon radiance, regions of interest (ROI) were selected on the surface of the mice or the 24-well plate using the automatic ROI. Bioluminescence was measured quantitatively by the Living Image® software, which gave the total flux of photons or radiance (photons/second from the surface) in each pixel, summed or integrated over the ROI area, in a square centimeter (cm2) of the tissue. These data were multiplied by one steradian (sr). The photon radiance is displayed as the average radiance, which is the sum of the radiance from each pixel inside the ROI/number of pixels or superpixels (photons/sec/cm2/sr) and the standard deviation of the pixel radiance inside the ROI. Bioluminescence, acquired by the CCD camera, was quantified by the Living Image® software using three types of ROI: (1) the automatic ROI measurement tool, which identifies bioluminescent emission automatically, considering a threshold of 20 to 28%; (2) the average background ROI, which measures the background signal in the area specified by the user and corrects the bioluminescent emission by subtraction; and (3) the subject ROI, which identifies each animal in a image. Whenever necessary, individual bioluminescent spots (foci) were summed to get the photon radiance measurements (photons/sec/cm2/sr) of each region of the animal: abdomen, thorax, paws, snout, ears, and urogenital region.
In the plate, bioluminescence acquired by the CCD camera was obtained with two types of ROI, the automatic measurement ROI, using a threshold of 25%, and the average background ROI. After subtraction of background signal, bioluminescent radiance measurements (photons/sec/cm2/sr) of spots distributed in the organ were summed to get the total radiance measurement of each organ: heart, rectum, gonads, fat tissue, intestines, tail, ears, snout, hind limbs, and forelimbs. Thereafter, the sum of bioluminescent radiance found in the whole 24-well plate, considered 100%, was used to estimate the percentage of bioluminescence in each organ.
Mice infected with Dm28c-luc, with Dm28c-WT and uninfected control mice, were euthanized at the time points indicated in the figure legends to collect heart, rectum, gonads, fat tissue from gonadal depot, intestines, tail, ears, snout, hind limbs, and forelimbs to be processed as described elsewhere . Briefly, fragments were fixed using Millonig-Rosman solution , and 5 μm-thick slices of paraffin-embedded samples were further processed and stained using hematoxylin-eosin (HE). To obtain different and representative regions of tissues, two 5 μm-thick slices in an interval of 80 μm were collected per slide. Qualitative analysis of tissues was based on cellular inflammatory infiltration and parasite nests.
Blood from mice infected with Dm28c-luc, with Dm28c-WT and uninfected control mice, was obtained by cardiac puncture using 3.8% sodium citrate and plasma was separated after centrifugation, 1000 g/10 minutes at 4°C, for cytokine analysis by flow cytometry (BD FACSCalibur™ flow cytometer) using the Cytometric Array kit (FlowCytomix Mouse Th1/Th2 10plex) (eBiosciences, San Diego, CA). This is a bead-based detection system for quantitative detection of GM-CSF, IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, and TNF-α. The evaluation was carried out according to manufacturer instructions. Statistical analyses were performed using the Excel software. Data from cytokine analyses of Dm28c-WT and Dm28c-luc were compared using a two-tailed unpaired t- test. Differences were considered significant if p values were < 0.05.
Results and discussion
Longitudinal evaluation of T. cruzi infection in mice
We showed in a previous publication that the genetically modified bioluminescent epimastigote forms of Dm28c-luc were stable after two years of cultivation in medium and were able to differentiate into metacyclic trypomastigote forms that infected LLC-MK2 cells in vitro. Transgenic trypomastigote forms of Dm28c-luc, derived from cell culture, showed stable expression for six months (the period tested) , allowing longitudinal evaluation of the acute and chronic phases of infected mice.
As a first step to characterize infection in mice using transgenic Dm28c-luc parasites, we compared blood parasitemia with that of mice infected with the Dm 28c-WT. As shown in Figure 1B, mice infected with the wild type strain showed a typical parasitemia curve for Dm28c, which increased up to approximately 20 dpi (3.0 ± 1.0 × 105 trypomastigotes/mL) and then gradually decreased. Parasitemia peak and magnitude is in accordance with previous publications [22, 23]. However, in mice infected with Dm28c-luc trypomastigote forms we found lower levels of parasitemia (Figure 1), but those mice showed active infection with patent levels of circulating parasites at all time points. This observation indicates that the course of infection may vary between Dm28c-WT and Dm28c-luc parasites and indicates that caution may be taken in the interpretation of results obtained with transgenic parasites.
In vivo and ex vivo evaluation of acute and chronic phases of T. cruzi infection
In vivo evaluation on the third week (21 dpi) demonstrated that bioluminescence was reduced in the thorax, snout, ears, paws, and abdomen (Figure 5E–all panels; Figures 6A and B), but the urogenital region displayed stable bioluminescence (Figure 6A). Ex vivo evaluation of infected organs by bioluminescent imaging demonstrated that the colon, cecum, and rectum displayed patches of bioluminescence that corresponded to 60% of bioluminescence found in the organs investigated in the plate (Figure 7B; Figure 8B), followed by the testicles and tail, which displayed 20% of bioluminescent emission; the heart showed 3% (Figure 8B). On the fourth week (28 dpi) post inoculation, bioluminescence declined overall in the body (Figure 5F–all panels, Figure 6A). Ex vivo evaluation revealed that infection was mainly distributed along the intestines and rectum, followed by the gonads and tail (Figure 7C, Figure 8C). Chronically infected mice, 180 dpi, displayed some foci of infection in the urogenital region, which could be quantified (Figure 6A and Figure 7E–all panels). To show that bioluminescent emission is directly related to mice infected with Dm28c-luc, images of mice infected with Dm28c-WT were included as negative control (Figure 7F). Ex vivo evaluation of dissected mice corroborated that bioluminescence was predominant in the intestine, followed by the rectum and tail (Figure 7D, Figure 8D). In other organs, such as the heart, bioluminescence was undetected. Therefore, the ex vivo evaluation supports the analysis made in vivo and shows the precise sites of infection in the urogenital region. The same profile of infection, with distribution of bioluminescent parasites for some regions of the body and its containment to the urogenital region, was observed in BALB/c and Swiss mice (data not shown).
Genetically modified Dm28c-luc versus Dm28c-WT strain
Independent of strain tropism, previous publications demonstrated a decline in intestinal mobility, evaluated by charcoal elimination, in mice infected with different T. cruzi strains, such as Dm28c, CL-Brener, Brazil, and TcY. In the same work, it was demonstrated that Dm28c interferes with intestinal mobility earlier than do other strains, in the first week of infection .
In our study, the alterations observed in tissues of rectum and anal sphincter are compatible with an inflammatory process that could lead to disruption of the nervous system; however, other tissues, glands, and regions of the intestine (not investigated) could also be sites of infection, as observed with naturally infected Opossum. Thus, the novel foci of infection in the acute and chronic phases, such as (a) the rectum, (b) the anal sphincter, (c) the testicle and its associated adipocyte tissues, and (d) the base of the tail, could also have an impact in the maintenance of infection in sylvatic, peri-domestic, and domestic cycle and in the transmission of the disease by routes not explored previously. In the case of Opossum, contamination of environment and food with parasites from the anal gland region has been shown . Adipose tissue was described as playing a role as a reservoir for recrudescence of infection .
Considering that the heart is one of the most studied and affected organ by T.cruzi infection, we performed also an ex vivo evaluation of the heart by bioluminescent imaging coupled to histological analysis, and compared the myocardium infection caused by Dm28c-WT and Dm28c-luc. In mice infected with Dm28-luc, bioluminescent emission could be detected until 28 dpi with a peak at 14 dpi, and by histological analysis a few amastigote nests and scattered inflammatory cell infiltrates could be observed at 14 dpi (Figure 9I and J). However, in mice infected with Dm28c-WT, heart tissues showed more amastigote nests and a few inflammatory infiltrates (Figure 9L). In the third week after infection (21 dpi), a few and small inflammatory infiltrates dispersed in the cardiac fiber of mice infected with Dm28c-luc were observed, some of them with amastigotes (Figure 9K). However, in mice infected with Dm28c-WT a few but clearly defined amastigote nests and inflammatory infiltrate were observed (Figure 9M). At 28 dpi no amastigote nest and inflammatory infiltrate was found, only discrete and scattered inflammatory cells were observed in mice infected with Dm28c-WT or Dm28c-luc (Figure 9N).
In the murine model, previous studies have demonstrated the evolution of experimental T. cruzi infection in heart tissues, with the presence of extracellular matrix components and inflammatory infiltrate in the myocardium, caused by Y strain in the acute phase of the disease. In opposition, infection caused by Dm28c strain failed to display inflammatory infiltrate and showed rare amastigote nests , which suggested that different strains could display different heart tissue responses [27, 28].
In order to find other sites of parasite reservoirs and infection, we evaluated tissues from the snout region such as the cheeks and nose of infected mice by histopathology analysis. In accordance with bioluminescent imaging, on 14 dpi, parasite nests were also observed in the snout, in muscle fibers of mice infected with each strain, Dm28c-WT (Figure 9O) or with Dm28c-luc (Figure 9P). Considering the organization of the tissue, which was composed of collagen and connective tissues, it was more difficult to evaluate the size of nests and to find parasite nests on 21 and 28 dpi. It is important to point out that in spite of the small size of amastigote nests in the muscle fibers of the snout, bioluminescent emission was above the threshold and displayed enough photons to be captured and quantified, which confirms that firefly luciferase expression in Dm28c-luc is stable and suitable for bioluminescent image studies.
Although amastigote nests were considerably more frequent in animals infected with Dm28c-WT, the Dm28c-luc strain was able to infect mice and displayed the same pattern of infection. In vivo and ex vivo bioluminescent imaging associated with histological analyses of tissue sections corroborated that amastigote nests were larger and more frequent in the rectum and anal region, if compared to the nests found in the heart and other tissues, and these sites of infection remained during the chronic phase.
Another aspect that should be considered about bioluminescent imaging is that the methodology is able to identify even small foci of infection in the organ, but depending on the level of luciferase expression and the tissues infected, the bioluminescent signal can be under the detection limit. Due to the limitations of histological techniques to evaluate residual and small foci of infections in tissues and organs, additional techniques such as PCR and qPCR should be considered to evaluate the presence of parasites in tissues and correlate bioluminescent image with parasite burden.
Previous work has shown the use of bioluminescent imaging in vivo to follow the parasite expressing firefly luciferase within their mammalian host , and developed a high-throughput bioluminescent imaging assay for drug trials in mice inoculated with T. cruzi in the hind footpads . It was observed that the progression of infection in mice infected with CL strain by intra peritoneal inoculation was similar to our study, displaying a peak at 10 to 14 dpi, but ex vivo bioluminescent imaging was restricted to a few organs . Thus, we performed longitudinal evaluation with quantification of bioluminescent foci in vivo coupled to a detailed ex vivo analyses of several regions and organs until the chronic stage of infection, 180 dpi. Our study gives new information and insights about the progression and distribution of infection throughout tissues and foci that are not usually explored but are important sites of infection, and should be considered in studies with murine models. If this pattern of infection is also related to the strain Dm28c or to the site of inoculation, it is the scope of our next work.
The biological and genetic variability of T. cruzi has led to its classification into six groups or discrete typing units (DTU) [31, 32]. In this context, Dm28c is classified as TcI; this strain was isolated from Didelphis marsupialis in Venezuela, and the TcI genotype is widespread in Colombia, Venezuela, and Central America, displaying emerging importance [33, 34]. Genotype analysis of isolates from chagasic patients, domestic and wild mammals, and triatomine bugs in Venezuela demonstrated that this group of T. cruzi accounted for most of the isolates . However, the distribution of infection throughout the new foci of infection such as, (a) the rectum, (b) the anal sphincter, (c) testicles and (d) its fat tissues, is usually not evaluated in the population affected by Chagas disease and during treatment, but can have a role in disease prognosis.
The present work demonstrated that bioluminescent imaging associated with microscopic examination of tissue sections is a powerful technology that can be used for evaluation of infection in the murine model.
The genetically modified strain is stable, and suitable for follow-up studies in vivo in the murine model, whose intensity of bioluminescent emission increases in accordance with the inoculum used to infect—106 (Figures 3A and B) or 105 (Figures 5A and B). The fact that the transgenic strain could be less lethal, but able to evolve into the chronic phase for 6 months to one year (not shown), can be an advantage for drug trials in vivo. This methodology brought, for the first time, new information about the progression and dynamics of infection, detecting new sites of infection.
We are grateful to Cynthia M. Cascabulho, from Instituto Oswaldo Cruz- IOC/ FIOCRUZ - Plataforma de Citometria de Fluxo, and Heliomar P. Marcos from Hospital Universitário Clementino Fraga Filho-HUCFF/UFRJ, for their assistance with flow cytometry and histological techniques. This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Financiadora de Estudos e Projetos (FINEP), Fundação de Aperfeiçoamento de pessoal de Nível Superior, and Fundação Carlos Chagas Filho de Amaparo à Pesquisa Científica do Estado do Rio de Janeiro (FAPERJ).
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