The authors thank Amanda Franscisco, John Kelly, and Fanny Escudi&#233; for providing BLI training and support on drug efficacy assays, John Kelly and Simone Calderano for providing parasites, and Gabriel Padilla for support with animal studies. A.C.S received a CAPES PSDE Scholarship for training at the London School of Hygiene and Tropical Medicine (United Kingdom). The authors also would like to thank the Flow Cytometry and Imaging Research (FLUIR) Platform at the Core Facility for Scientific Research - University of Sao Paulo (CEFAP-USP) for technical support with the IVIS Spectrum equipment analysis, and the Laboratory of Genetics and Sanitary Control ICB-USP for the post-experimentation assays for quality control of mice as specific pathogen-free. This project was funded by DNDi. DNDi is grateful to its donors, public and private, who have provided funding for all DNDi activities since its inception in 2003. A full list of DNDi&#39;s donors can be found at&#160;https://dndi.org/about/donors/.

In Vivo Bioluminescent Model of Chagas Disease for Drug Evaluation

Authors have no conflicts of interest to declare.

Bioluminescence imaging is a breakthrough method that allows the detection of a report gene using a visible and infrared spectrum of electromagnetic radiation. Therefore, there is no need for radiolabeled markers to trace your specimen35. BLI is suitable for rodent models and other small species. It is very useful for preclinical studies because it is safer and allows several image rounds, causing minimal animal discomfort. Besides, in vivo imaging is very flexible due to the possibility of combining bioluminescence, fluorescence, and other techniques such as positron emission tomography36.
Optical imaging is ruled by optical physical properties, like absorption and scattering. All tissues absorb and scatter light of distinguished wavelengths differently37. One critical step is selecting a reporter gene without taking into account the emitted wavelength of the light produced by the chemical reaction. While a reporter gene expression level may be high in vitro in bioluminescent assays, the same levels of expression may not be achieved when progressing to the in vivo setting. In this protocol, we used red-shifted Photinus pyralis luciferase (PpyRE9H)38 codon-optimized version for trypanosomatids9, which emits light at 617 nm, one of the most suitable for in vivo studies39. Wavelengths longer than 600 nm are less absorbed and scattered by body endogenous chromophores, especially hemoglobin and melanin. Thus, red lights can be transmitted through several centimeters of tissue, allowing the photons to reach the CCD camera even from within visceral tissues39,40.
One area of concern in imaging settings is the lack of a comprehensive understanding of their function and effects. Binning, a pre-processing technique, combines the information acquired by contiguous detectors into a larger pixel. This process enhances the signal-to-noise ratio, reducing background noise and improving sensitivity. However, it decreases spatial resolution accuracy, resulting in a pixelated image41,42.&#160;This trade-off is an important consideration in your imaging strategy.
Based on the Target Product Profile and Target Candidate Profile for Chagas disease34, the proof-of-concept study is focused on sensitivity to detect T. cruzi and help establish if a new drug candidate can achieve a sterile cure (represented by lack of relapse after several immunosuppression rounds). Therefore, we execute the BLI using the highest binning factor without supersaturating the image. When the image supersaturates, a new acquisition is performed using a lower binning factor. During the analysis, a mathematical correction is applied to the images that required different binning. This way, the final data should be presented using the same binning. Table 1 demonstrates the different values obtained when distinctive binning factors were applied in the same image and ROIs.
Table 1: Influence of binning settings on the BLI quantification. Quantification of three ROIs on the image of acute model (d13) and chronic model (d118), analyzed in different binning factors. <a href="https://www.jove.com/files/ftp_upload/66740/DaSilvaetal_Table1_Reviewed.xlsx" target="_blank">Please click here to download this Table.</a>
Due to the current scenario of Chagas disease in the clinic, drug discovery efforts aim to completely eliminate parasites&#160;(parasitological cure)27,34. Therefore, the in vivo preclinical protocol includes approaches that overcome the limitations of BLI technical sensibility. One of the approaches is treating the mice with cyclophosphamide to decrease the immune response that controls the parasite load. Another strategy is diminishing the tissue deepness and removing layers of muscle, skin, and fur that obstruct the light path to the camera. Through the ex vivo procedure, small bioluminescent spots can be detected, revealing parasite foci below the&#160;in vivo BLI threshold, as shown in Figure 5C in ex vivo result of mouse treated by Posa.
Designing a pilot experiment to evaluate the model itself and the infection dynamics is crucial to establishing an accurate experiment for antiparasitic drug efficacy assessment. Hence, the researcher will be able to define the proper BLI settings and infection time course in advance. In an exploratory experiment, one tool that can be helpful to define the acquisition settings is the &#39;Autoexposure&#39;. With this tool, the researcher establishes the priority of three settings (Exposure time, Binning, and F/Stop) to acquire the best image possible. In particular, the researcher should ensure that images are acquired within the dynamic range of the CCD camera, without supersaturation or underexposure, which can be checked through the minimum and maximum limits of the scale and the autoexposure feature (Menu Edit &#62; Preferences &#62; Tab Acquisition &#62; Tab Autoexposure). In this protocol, the camera aperture was set at the maximum value (F/Stop: 1), and different exposure times and binning factors were defined for acute and chronic models. These settings give time predictability to perform different image rounds simultaneously. Considering the reporter method is based on an enzymatic reaction, both the biodistribution of the substrate into the mouse and the luciferase kinetics influence the bioluminescent signal and, thus, infection quantification (Figure 1B). Consequently, acquiring images in different moments of enzymatic kinetics introduces data variability that cannot be accounted for or corrected and impacts the total flux (photons/second) or radiance (photons/second/cm2/steradian) calculation. Besides, T. cruzi infection displays dynamic spatial positioning in mice (different areas and tissues, deepness, and parasite load). Hence, establishing a value of counts to acquire could miss weaker signal sources (low number of parasites in a certain spot deeper in the tissue) if another stronger signal&#39;s source meets the defined auto-exposure criteria.
A tricky feature of Living Image software is displaying the acquired image in an automatic color scale. There is no option to pre-set the scale to exhibit automatically a brand-new acquired image according to the selected scale values (see protocol step 6.2). This situation forces the researcher to manually change the images one by one to the chosen max and min values. As a consequence, the non-experienced and not well-trained users do not have the proper readout during the acquisition session, and they could mislead the data or lose important information at that time point. For that, the pilot experiment is beneficial.
One of the most common questions about the proof-of-concept experiment design is how to choose the treatment duration and dose. For new chemical entities, these parameters are usually defined by the compound potency and selectivity in vitro, in combination with data generated by drug metabolism and pharmacokinetics (DMPK) and tolerability studies conducted prior to testing in vivo for efficacy. In summary, after identifying compounds that are able to selectively kill the parasite inside cells, the first ADME experiments (absorption, distribution, metabolism and excretion) are performed in vitro to estimate compounds&#39; aqueous solubility, cell permeability and metabolic stability, among other parameters. If compounds show a good balance of in vitro properties (usually defined in target candidate profiles), then these candidates are progressed to in vivo pharmacokinetics (PK) studies in healthy mice, which outline the compound exposure in the blood (and possibly in tissues as well) and provide a general idea of the tolerability at different dose levels17,34,43. Ideally, the goal of the PK assessment in most infectious diseases is to determine the feasibility to reach free plasma concentrations (corrected for plasma protein binding) that are above the EC50/EC90 concentrations44 -&#160;the effective concentration that kills or at least inhibits the growth of 50% or 90 % of parasites, respectively - for a sufficiently long period of time. If enough exposure is achieved at a certain dose level, then this regimen can used during the efficacy studies using the Chagas BLI model. For drug repositioning studies, in vitro and in vivo PK data should be available. A good start for drug reprofiling is chemical databases such as PubChem45, which provide recognized data that can be converted to mice using allometric scaling46 to estimate safe and non-toxic treatment regimens to be tested. However, that is not always the case. PK studies are still an overlooked field in academic science, and few pharma companies publish their PK results. The drug discovery science community recommends including in vivo PK assessment along with in drug efficacy assays (pharmacodynamics)47. Therefore, preclinical imaging is compatible with compound measurements simultaneously, and this associated approach enhances data robustness.
In addition, the mice&#39;s handling, weight, and health conditions are monitored throughout the entire experiment. Signs of toxicity and side effects such as hunching, shaking, loss of balance, unwillingness to move, reluctance to feed or drink, prostration, or any other abnormalities present in the group or by individual mouse conditions should be registered and reported in preclinical studies. One of the goals of optical imaging is to ensure the animals&#39; well-being. Thus, humane endpoints should be applied in mice with pain signs described in the &#39;Grimace scale48. Also, mice were&#160;weighed weekly&#160;during BLI acquisition and, more often, during drug dosing and CTX treatment. Following animal welfare regulations, mice that lose more than 20% of body weight must be immediately humanely euthanized.
The T. cruzi bioluminescent model is now the state-of-the-art experimental model for the discovering&#160;and developing&#160;of new treatments for Chagas disease. A model that replicates key features of T. cruzi infection and Chagas disease49, allowing for real-time monitoring of parasitemia and differentiation of compounds with varied efficacy profiles associated with known modes-of-action. BLI is a technique that enhances assertiveness in identifying infected tissues. It enables the precise selection of infected tissues to be used in a broad range of approaches, including all classic methods already applied in T. cruzi research50,51. Additionally, it allows researchers to explore cutting-edge technologies and develop new ones33. In addition, BLI provides improvement of animal well-being and more rational use according to 3Rs principles10,35, all at once.
Several research groups focus on neglected tropical diseases are placed in countries where in vivo imaging devices are unavailable. To overcome the current scenario, new international networks like Global BioImaging and their associated consortia promote actions to provide open access to imaging core facilities and improve staff and imaging scientists&#39; training52,53. These initiatives, along with friendly user protocols like this one, can afford conditions democratizing high-end technologies for all researchers. The implementation of this method in the preclinical drug discovery offered a solid efficacy readout and predictive value of clinical outcome facilitating drug discovery for Chagas disease.

Chagas disease is endemic in Latin America and affects approximately seven million people worldwide1. Annually, more than 50,000 deaths and economic losses of around 7 billion dollars&#160;result from the disabling nature of this disease2. Chagas disease is caused by the protozoan Trypanosoma cruzi, a heteroxenic hemoflagellate parasite capable of infecting mammals (wild and domestic) and triatomine vectors (Hemiptera, Reduviidae)3 in the Americas, where the vectorial transmission is established. Other important infection routes include blood transfusion, organ transplantation, oral (by the ingestion of food contaminated with an infected triatomine)4, and congenital transmission. Non-vectorial transmission routes have contributed to the spread of Chagas disease to non-endemic areas3,5.
Chagas disease manifests in two clinical phases. The acute phase is, in most cases, asymptomatic. Symptomatic infections are usually associated with non-specific signs such as fever, fatigue, myalgia, lymphadenopathy, splenomegaly, and hepatomegaly. The acute phase is also often associated with patent parasitemia and systemic circulation of parasites. Death may occur in up to 10% of the diagnosed cases, especially in those of oral infection6. The chronic phase is often characterized by a long period of absence of any symptoms. With time, approximately one-third of the patients infected decades earlier exhibit cardiac manifestations, usually accompanied by fibrosis and myocardial inflammation, and/or gastrointestinal disorders mostly related to the development of megaesophagus and/or megacolon syndromes3,5,6.
The etiological treatment of Chagas disease consists of only two drugs: benznidazole and nifurtimox. These antiparasitic agents have been available for over 50 years and have considerable toxicity and limited efficacy5,7,8. Consequently, there is a pressing need to develop new, safe, and more efficacious treatments for Chagas disease patients.
More sophisticated and accurate techniques make it possible now to obtain answers to old questions that allow advances in the search for new treatments for Chagas disease. In this sense, the scientific community greatly benefits from genetically modified parasites for in vivo studies on the course of infection and evaluation of drug efficacy9,10,11,12. A longitudinal assay based on the bioluminescence imaging (BLI) system allows efficacy evaluation during and after the treatment regimen, leading to the identification of compounds with trypanocidal activity10,13. The BLI method provides a direct measurement of parasite load, both in circulation or in tissues and organs, through the quantitation of light produced by the genetically modified T. cruzi CL Brener Luc::Neon lineage11, which constitutively expresses the red-shifted firefly luciferase12.
Nonetheless, nearly 10 years after the establishment of the Chagas disease BLI animal model and drug efficacy studies, only a few research groups dominate this technique. This fact is due to not only the reduced access to proper imaging equipment but also the lack of training and availability of structured, detailed protocols. This method presents several advantages over other approaches, which rely on the assessment of parasitemia by microscopy, serology, or organ/tissue infection evaluation by qPCR for parasite DNA detection, as it allows improved mice well-being and reduction of animal use by the possibility of generation of more robust and integrated data in vivo. Furthermore, this method is, arguably, more sensitive, as it enables ready detection of parasite foci in visceral organs after drug treatment10,12. Therefore, this protocol aims to guide research groups on Parasitology and other infectious diseases to establish this methodology in their laboratories by detailing the technical procedures. Here, we share the experience obtained through implementing of the Chagas disease BLI model in Brazil, the first of its kind in Latin America, as part of the drug discovery efforts coordinated by the Drugs for Neglected Diseases initiative (DNDi).

<table><tbody><tr><td>BD LSRFortessa&amp;trade; X-20 Cell Analyzer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Weighing Balance (animal facility)</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>Revvity (former PerkinElmer)</td><td></td><td></td></tr><tr><td>FlowJ Software v10.7.1</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Living Image Software for Spectrum v4.7.1</td><td>Revvity (former PerkinElmer)</td><td></td><td>License Free Analysis Software called &#039;Aura Imaging&#039; could be used for the most basic features provided by Spectral Instruments Imaging (Bruker company) (https://spectralinvivo.com/software/)</td></tr><tr><td>Microsoft Office software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>GraphPad Prism v8.4.0</td><td>GraphPad Software Inc.</td><td></td><td></td></tr><tr><td>DMEM Low Glucose</td><td>Vitrocell</td><td>D0025</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Foetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Trypsin 0.5% EDTA</td><td>Gibco</td><td>25300-062</td><td></td></tr><tr><td>LIT medium</td><td>In house</td><td></td><td></td></tr><tr><td>Hygromycin B (50 mg/mL)</td><td>Gibco</td><td>10687010</td><td></td></tr><tr><td>Grace&amp;prime;s Insect Medium</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G9771</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich&amp;nbsp;</td><td>54457</td><td></td></tr><tr><td>IVISBrite d-luciferin potassium salt</td><td>Revvity (former PerkinElmer)</td><td>122799</td><td>Also could be used: VivoGlo Luciferin, in vivo grade (Promega/P1043); D-Luciferin, Monopotassium Salt (Thermo Scientific/88293) or PierceD-Luciferin, Monosodium* Salt (Thermo Scientific/88291); D-Luciferin, Potassium Salt (GoldBio/LUCK or eLUCK); D-Luciferin, Sodium* Salt (GoldBio/LUCNA or eLUCNA) *Sodium or potassium salt differences relies minimal chances on solubility, however do not affect in vivo performance.&amp;nbsp;</td></tr><tr><td>DPBS</td><td>Gibco</td><td>21600-044</td><td></td></tr><tr><td>Cyclophosphamide (CTX)</td><td>Sigma-Aldrich</td><td>C0768-5g</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>(Hydroxypropyl)methyl cellulose (HPMC)</td><td>Sigma-Aldrich</td><td>09963-25G</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma-Aldrich</td><td>402834</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P1754-1L</td><td></td></tr><tr><td>Benznidazole</td><td>ELEA</td><td></td><td></td></tr><tr><td>Posaconazole (Noxafil commercial formulation)</td><td>Schering-Phough</td><td></td><td></td></tr><tr><td>Giemsa</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>gavage needle (stainless-steel straight) - 22GA</td><td>Aton</td><td>&amp;nbsp;CA2003</td><td></td></tr><tr><td>1 mL Syringe and 31G needle</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>1 mL Syringe and removable 26G needle</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>1 mL Syringe and removable 24G X&amp;frac34; needle</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>Sterile Syringe Filter 0.2 &amp;micro;m</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>A4 Matte Black paper 120gr or thicker</td><td>Paper Color/ Canson (Available from several suppliers)</td><td></td><td></td></tr><tr><td>aluminum foil</td><td>Available from several suppliers</td><td></td><td></td></tr><tr><td>Neubauer chamber</td><td>Available from several suppliers</td><td></td><td></td></tr></tbody></table>

demystifying in vivo bioluminescence imaging of a chagas disease mouse model for drug efficacy studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Using an adequate mouse model based on transgenic parasites that constitutively express luciferase, it is possible to reproduce key human T. cruzi infection features causing minimum harm to the host, allowing real-time tracking of the parasites by whole-body BLI of the host in a longitudinal (lifelong) manner, for up to several months12.
Figure 3 shows a pilot experiment&#160;demonstrating the CL Brener Luc::Neon infection time-course from day 1 post-infection until 150 dpi in BALB/c mice. In the first 20 dpi, bioluminescence-inferred parasite burden increases, typical of parasitemia peak, followed by a sharp decrease in parasite load, due to mice immune control. Thus, the acute phase of the infection is considered the first 50 dpi. The chronic phase is defined when infection reaches a constant bioluminescent rate, from 100-150 dpi.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66740/66740fig03v2.jpg" /> 
Figure 3: Understanding the model. Kinetics of Trypanosoma cruzi CL Brener Luc::Neon infection in BALB/c mice (n = 11). Top panel: ventral and dorsal bioluminescent images from different time points for 150 days post-infection (dpi) with 1 x 10&#179; bloodstream trypomastigotes by intraperitoneal injection. Heatmap scale (in Log10) of bioluminescent signal in radiance (p/s/cm2/sr). Color code: Purple = 5 x 103; red = 1 x 107. Bottom panel: bioluminescent signal quantification in Total Flux (p/s) of whole-body mice. Data are expressed as means (lines) and standard deviations (shaded regions). Periods defined for acute and chronic phases of infection in this mouse model are highlighted in orange. Luminescence mean values of non-infected mice (n = 3) are shown as the threshold (light gray line). <a href="https://www.jove.com/files/ftp_upload/66740/66740fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The therapeutic outcomes can also be predicted since the main phases of the infection are well-reproduced in a mouse model. Thus, BLI is now applied to translational science through proof-of-concept studies that support decision-making about compound efficacy potential and progression in the development pipeline10,27. In the acute mouse model, the oral treatment should start when the infection reaches the parasitemia peak (around 14-21 dpi), characterized by a high number of trypomastigotes in the bloodstream (approximately 1 x 105 trypomastigotes/mL - data not shown) and systemic infection. In the chronic phase, mice treatment starts at 100 dpi, when the parasite load is comparatively much lower, with stable subpatent parasitemia.
To demonstrate the application of the protocol described above in the evaluation of antiparasitic agents, we compared the treatment efficacy of benznidazole (BZ), the standard drug used for treatment of Chagas disease, and posaconazole (Posa), a sterol 14&#945;-demethylase (CYP51) inhibitor that failed in clinical trials for Chagas disease, and that has also been shown to be inefficacious as antiparasitic agent against T. cruzi in vitro and in vivo, including in the BLI model described in this protocol13,18,28,29,30.
For the acute mouse model, cohorts of 6 mice per group were treated orally by gavage31 for 20 days with Posa at 20 mg/kg or BZ at 100 mg/kg, once daily. Mice were also treated with BZ for 5 days at 100 mg/kg once daily to evaluate the effect of short treatments. After compound elimination (10 days after the treatment ended), the BLI-negative mice were immunosuppressed, a condition that promotes infection relapse when a parasitological cure is not achieved (Figure 4A). Posa treatment resulted in a 99.49% &#177; 0.27% (mean &#177; standard deviation) reduction in bioluminescence-inferred parasite burden at the end of the treatment and remained at similar levels of non-infected mice during the drug compound elimination period (except for mouse #3, with a transient signal at 40 dpi, and mouse #2, that demonstrated a weak BLI spot at 44 dpi). Compared to the non-treated group, BZ treatment for 20 days reduced 100% &#177; 0.01% the bioluminescence-inferred parasite burden at the end of the treatment. In contrast, the short treatment with BZ for 5 days led to a 99.98% &#177; 0.03% reduction at 19 dpi (similar to the threshold level). However, in this instance, the BLI reduction was transient and in the subsequent acquisitions, all mice presented infection reactivation (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66740/66740fig04v3.jpg" /> 
Figure 4: Proof-of-concept experiment design and results obtained with bioluminescence imaging in Chagas disease acute model. (A) Schematic chart of timeline process in acute Chagas disease mouse model for preclinical studies for assessment of compound efficacy. Black dots: imaging time points. Medicine flask and orange bar: treatment start and end, respectively. Syringe icon: cyclophosphamide injections. dpi: day post-infection. Color code: gray = non-treated infection; orange = treatment period; blue = compound elimination phase; yellow = immunosuppression period. (B) Time-course of treatment of Trypanosoma cruzi. Left panel: ventral bioluminescence images of BALB/c mice (i) non-treated or treated for 20 days with (ii) posaconazole (Posa) at 20 mg/kg, (iii) benznidazole (BZ) at 100 mg/kg treated for 20 days and (iv) BZ at 100 mg/kg treated for 5 days with (n = 6/group). All treatments were orally administered by gavage (10 mL/kg) once a day. Log10 scale heatmap indicates the bioluminescent signal intensity from low level (blue) to high (red). Right graph: whole-body quantification of bioluminescence imaging data. Data are expressed as means (lines) and standard deviations (shaded regions). Key: Medicine flask: treatment start (14 dpi); orange bar: treatment end (18 dpi/33 dpi); syringe icon cyclophosphamide injections (from day 44-53);&#160;orange dot: mouse analyzed by ex vivo procedure;&#160;&#167; data excluded due to D-luciferin leakage in urine. (C) Ex vivo procedure for T. cruzi foci detection in organs and tissues. Plate distribution key is presented at the bottom of the figure (created with BioRender.com: ZY26LG8AOF). Same radiance scale as shown in vivo panel. <a href="https://www.jove.com/files/ftp_upload/66740/66740fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this model, antiparasitic treatment may reduce parasite load below the BLI detection limit of 1000 parasites. Thus,&#160;mice appear as BLI negative10. To ensure that a parasitological cure has been achieved, it is necessary to promote immunosuppression of mice using cyclophosphamide treatment29,32. Mice that still have an undetectable parasite load after drug treatment will become BLI-positive after immunosuppression. This effect is shown more extensively by Posa treatment in the acute model, which promotes parasite relapse. In this experiment, the ex vivo procedure was performed on mice with the lowest bioluminescent signal to evaluate parasite tissue tropism (Figure 4C). Non-treated mice&#160;shows strong bioluminescent signals, especially in the gastrointestinal tract (TGI) and associated tissues such as visceral fat and mesentery. In addition, the skin was a site associated with parasite persistence. Mice treated with Posa presented lower-intensity bioluminescent spots that were more restricted to the colon, mesentery, and visceral fat. On the other hand, in mice treated with a curative treatment regimen, as BZ 100 mg/kg for 20 days, no bioluminescent signal is detected even under immunosuppression. Therefore, considering that after promoting conditions for bioluminescent signal rising above the threshold level of 1000 parasites10, and increasing sensitivity by exposing the visceral organs, it is considered that BZ 100 mg/kg for 20 days provides sterile cure. The assessment of sterile cure was previously validated by different techniques10,13,17,33,34.
A similar study design was applied to the chronic model (Figure 5A), in which mice (n = 6/group) started to be treated at day 100 post-infection. At this point, Posa 20 mg/kg, BZ at 100 mg/kg, or 10 mg/kg once daily were administered orally for 20 days. After the drug elimination period, the BLI-negative mice were immunosuppressed. In addition, to assess sterile cure, mice that were still BLI-negative at the end of the immunosuppression phase were subjected to ex vivo analysis.
In chronic infection, treatment with Posa at 20 mg/kg led to a decrease of 95.03% &#177; 6.18% in bioluminescence-inferred parasite burden at the end of the treatment and remained at similar levels of non-infected mice during the drug elimination period. After the immunosuppression, the majority of mice showed a variable level of BLI signal and distribution (Figure 5B). Ex vivo procedure revealed that one whole-body BLI-negative mice treated with Posa had a bioluminescent spot in the colon detectable via ex vivo examination (Figure 5C).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66740/66740fig05v3.jpg" /> 
Figure 5: Drug assessment experiment design and results achieved in the chronic model of Trypanosoma cruzi infection by bioluminescence imaging. (A) Schematic chart of experiment design of chronic Chagas disease mouse model. Black dots: imaging time points. Medicine flask and orange bar: treatment start and end, respectively. Syringe icon: cyclophosphamide injections. Dpi: day post-infection. Color code: Gray = non-treated infection; orange = treatment period; blue = compound elimination phase; yellow = immunosuppression; lilac = relapse. (B) Longitudinal evaluation of treatment efficacy in the Chagas disease chronic model. Left panel: ventral BLI of BALB/c mice (i) non-treated or treated for 20 days with (ii) posaconazole (Posa) at 20 mg/kg; (iii) benznidazole (BZ) at 100 mg/kg, and (iv) BZ at 10 mg/kg (n = 6/group). All treatments were orally administered by gavage once a day. Right graph: sum of ventral and dorsal total flux raw data. Key: medicine flask: treatment start (102 dpi); orange bar: treatment end (121 dpi); yellow bar and syringe icon: cyclophosphamide injections (from day 131-142). Orange dot: mouse analyzed by ex vivo procedure. ! mouse died during anesthesia. <img alt="Equation 1" src="/files/ftp_upload/66740/66740eq01.jpg" style="margin: auto;" /> Mouse displays abdominal abnormalities. Heatmap Scale (Log10) indicates the bioluminescence intensity in radiance unit from low level (3 &#215; 105 as blue) to high (1 &#215; 107 as red). (C) Ex vivo analysis of T. cruzi tropism in tissue. Bioluminescence detection of excised organs from immunosuppressed in vivo BLI-negative mice after treatment with Posa and BZ at 100 mg/kg or mice with the lowest signal in non-treated and BZ at 10 mg/kg groups. Plate distribution key is shown at the bottom of the figure (created with BioRender.com: ZY26LG8AOF). Radiance scale is equal to in vivo panel. <a href="https://www.jove.com/files/ftp_upload/66740/66740fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Treatment with BZ at 100 mg/kg reduced the bioluminescence to threshold levels (93.87% &#177; 2.14%) at&#160;122 dpi and forward until the end of the experiment. Considering ex vivo analysis, BZ achieved a cure rate of 100% (6/6 mice), as no mice presented a return of bioluminescent signal even when immunosuppressed and internal organs were examined. That means a lack of relapse. Nonetheless, the BZ treatment regimen with a lower concentration resulted in a slight reduction of bioluminescence (85.95% &#177; 18.43%) but no cure, as mice continued to show detectable bioluminescent signal foci at all subsequent time points. Thus, this model allows quantitative differentiation between inefficacious treatments (posaconazole and suboptimal treatment with benznidazole) and efficacious treatments (optimal dosing and treatment length with benznidazole).
Supplementary Figure 1: Preinfection analysis of T. cruzi population expression of luciferase by measurement of the reporter gene mNeonGreen fluorescence. Flow cytometry of mNeonGreen fluorescent protein constitutively expressed by the CL Brener Luc::Neon parasite. Normalized histogram of parasite number (Y-axis) by mNeonGreen fluorescence intensity (X-axis). Internal legend demonstrates the analyzed strain and form, fluorescence median, and percentage of fluorescence population. Gate lines are defined by of CL Brener wild type (WT) strain as non-fluorescent control. <a href="https://www.jove.com/files/ftp_upload/66740/daSilvaetal_Supplemental_Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Example of analysis table in BLI measurement. Raw data obtained from bioluminescence imaging quantification of day 19 post-infection in the acute model&#160;used in the representative results shown in Figure 4B. Groups description: Control as non-infected mice (n = 3/group); vehicle as non-treated infected mice; posaconazole (Posa) at 20 mg/kg for 20 days; benznidazole (BZ) at 100 mg/kg for 20 days; BZ at 100 mg/kg treated for 5 days (n = 6/group). <a href="https://www.jove.com/files/ftp_upload/66740/daSilvaetal_Supplemental_Table1.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Real-Time Monitoring of Parasite Burden and Host Response

The protocol provided here describes the detailed steps to perform drug efficacy studies in a bioluminescent model of Trypanosoma cruzi infection, with a focus on data acquisition, analysis, and interpretation. Troubleshooting and quality control procedures to minimize technical issues are also provided.

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, ...

demystifying-vivo-bioluminescence-imaging-chagas-disease-mouse-model

Research

JoVE Journal

Immunology and Infection

1.4K Views.  Universidade de São Paulo. Onze onderzoeksgroep houdt zich bezig met het ontdekken van geneesmiddelen voor de ziekte van Chagas. We zijn op zoek naar nieuwe en betere medicijnen en proberen ook te begrijpen hoe de parasiet, Trypanosoma cruzi, in de gastheer kan blijven bestaan en zelfs na effectieve medicamenteuze behandeling kan overleven zonder resistentie tegen geneesmiddelen te ontwikkelen. De ziekte van Chagas is een complexe systemische infectie die parasitologische genezing vere...

Video: Auteur in de schijnwerpers: real-time monitoring van parasietenlast en gastheerrespons

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

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.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

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.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease 1, 2. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth 3. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing 3, 4. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation 3, 5-7, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1 8. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8&gamma;&delta;+, CD8&alpha; lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

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