The authors thank the Cirillo laboratory members for valuable discussions and assistance throughout this study. We thank Dr. Joshua Hill and the laboratory of Dr. James Samuel for assistance with during the filming of this protocol. This work was funded by grant 48523 from the Bill &amp; Melinda Gates Foundation and grant AI47866 from the National Institutes of Health.

1. Pulmonary Infection by Intratracheal Intubation
<ol>
<li>Weigh mice and, optionally, marks can be made on the ears for easy identification.</li>
<li>Anesthetize the mice with ketamine (100 &mu;g per g of mouse weight) and xylazine (10 &mu;g per g of mouse weight) by intraperitoneal inoculation.</li>
<li>Place mice in cages until fully anesthetized. Squeeze the pads of their feet to check pedal reflex. Mice should display reduced or no reflex reaction.</li>
<li>Place the mouse on the intubation stand lying on its back.</li>
<li>Fix a rubber band to intubation stand and then place it under the upper incisors of the mouse. Use tape to fix legs and arms onto the intubation stand.</li>
<li>Gently move tougue out of the mouth using forceps.</li>
<li>Insert speculum with octoscope inside of the mouth and look into oropharynx until larynx opening is visible.</li>
<li>Advance guide wire covered with cathether into trachea until catether hub is at the incisors. Remove guide wire. Put air into cathether to observe chest expansion and confirm correct intubation.</li>
<li>Inject 50 &mu;L of bacterial (in the images shown we used bacillus Calmette Guerin (BCG) expressing click beetle red luciferase (CBRLuc), constructed in our laboratory, but any luminescent bacterial strain can be used) solution through cathether using a 1 mL syringe.</li>
<li>Place mouse in a cage and observe it during recovery from anesthesia.</li>
</ol>
2. Animal Anesthetization and Preparation for Bioluminescence Imaging
Mice are anesthetized with isoflurane using the XGI-8 Gas Aneshesia System. 
<ol>
<li>Before operating the XGI-8 anesthesia system, weigh each charcoal filter canister and write the weight and date on it. If the weight is 50 gram over of the initial weight, replace it with a new canister.</li>
<li>Check the isoflurane level in the vaporizer and fill it if necessary.</li>
<li>Turn on the evacuation pump in the front of the XGI-8 anesthesia system and set to 8 lpm (liters per minuite).</li>
<li>Turn on the oxygen supply from the high pressure cylinder and set it to 55 psig.</li>
<li>Turn on the oxygen toggle (green) on the front of the XGI-8 anesthesia system.</li>
<li>Turn on the gas flow to the imaging chamber to set the level of gas flow. Set to 0.25 lpm and then turn off the gas flow.</li>
<li>Turn on the gas flow to the anesthesia induction chamber to set the level of gas flow. Set to 1.5 lpm and then turn off gas flow.</li>
<li>Turn on the isoflurane with the vaporizer and set to 2-2.5 percent. The isoflurane level can be adjusted depending upon the number of animals being used and weight.</li>
<li>Place mice into the induction chamber and close the lid. Turn on the gas flow to induction chamber. Place mice in the chamber for 5 - 10 minuites until they are anesthetize completely.</li>
<li>Apply optical ointment to the eyes to protect the mouse eyes during imaging and place the mice in the imaging chamber in one of the nose cones on the anesthesia manifold. Use light baffle between mice to prevent the reflection of light onto adjacent animal subjects.</li>
<li>Inject luciferin (150 &mu;g/gram of body weight) by intraperitoneal inoculation.</li>
</ol>
3. Bioluminescence Imaging
<ol>
<li>Start the Living Imaging Software.</li>
<li>If the system is not initialized, initialize the IVIS Imaging system.</li>
<li>Set the parameters for imaging by clicking sequence setup in IVIS acquisition control panel. 
Select luminescence and photograph in Imaging Mode. If the option to carry out DLIT 3D reconstruction is desired, select photographic, luminescent and structural light images as part of the image sequence. 
Set exposure time from 0.5 sec to 10 minutes. 
Set Binning and F/stop based on expected brightness of sample. 
Set excitation filter to block and emission filter to open, unless planning to acquire only specific wavelengths of light. In the case of DLIT 3D constitution, set multiple emission filters of different wavelengths to allow accurate localization of source. 
Set FOV from A to D depending on the number of mice or region of animal to be imaged. Select A and B for 1 mouse, C for 2-3 mice, and D for 4-5 mice.</li>
<li>Click add in the Image Wizard in the Acquisition Control Panel to add the sequence setup.</li>
<li>Start imaging sequence by clicking acquire. 
In the case of DLIT 3D construction, luminescent images will be acquired for multiple emission filters at different wavelengths. Photographic and structured light images will also be acquired.</li>
<li>During acquisition the image and edit image levels panels will be visible. Fill in as detailed information as possible for each experiment in edit image to ensure easy tracking of images at later times and save your images.</li>
<li>ReRemove the mice from IVIS imaging chamber and return them to their cages. Observe recovery from anesthesia to ensure mice were not significantly affected by the procedure. Animals should be monitored constantly until they fully recover from anesthesia (usually 1-2 min).</li>
</ol>
4. ex vivo Imaging and CFU Analysis for the Quantification of Bacteria in the Lungs
<ol>
<li>Inject mice with luciferin intraperitoneally in the same manner as for imaging, just prior to euthanasia.</li>
<li>Euthanize the mice by intraperitoneal injection of 100 mg/kg pentobarbitol 5 min after luciferin injection.</li>
<li>Explant the lungs from the mice and place in sterile petri-dishes using sterile forceps and scissors.</li>
<li>Place petri-dish containing explanted organ in imaging chamber and acquire bioluminescence images in the same manner as for whole mouse.</li>
<li>After imaging, remove explanted organ from petri dish using sterile forceps and homogenize in 1 mL PBS.</li>
<li>Make dilutions and plate tissue on the appropriate selective media. Homogenized tissue can be stored -80&deg;C for re-plating should it be necessary. Alternatively, homogenized tissue can be used for RNA or DNA extraction to quantify bacterial numbers by qPCR.</li>
</ol>
5. Analysis of Luminescence Imaging
<ol>
<li>Start "Living Imaging Software" and browse the image files.</li>
<li>Use "Tool Palette' to adjust images. Click Image Adjust to modify correction/filtering settings and min/max for individual color scales.</li>
<li>Use ROI tools in pulldown list for the quantification of light intensity. Select ROI shape, number and size. Drag ROI frame to region of interest on image. Ensure all ROI are the same size and shape. Click ROI measurement and save, copy and/or export quantitative data.</li>
<li>In the case of DLIT 3D reconstructions, load image sequence including the structured light image. Click Surface topography in the Tool Platte. In pulldown list, click Reconstruct tab to generate the surface topography. A surface mess will then appear in the window.</li>
<li>Select DLIT 3D reconstruction in the Tool Platte. In pulldown list, click the Properties tab to set tissue properties and source spectrum. Muscle is recommended under most circumstances. In the pulldown list, click the Analyze tab to select the wavelength for analysis. Click the Parameter tab and confirm default values or adjust DLIT parameters if necessary. Click Reconstruct tab to start 3D analysis.</li>
<li>When 3D reconstruction is finished, the 3D view will display the results of 3D reconstruction. Click results tab to see analysis data for photon density, voxels and DLIT algorithm parameters.</li>
<li>Save and/or export the results of the 3D reconstruction analyses into figures and data files.</li>
</ol>
6. Representative Results:
The bioluminescence images of mice infected with bioluminescent bacteria along with an uninfected mouse control are shown in Figure 1. The mice pulmonary infected with bioluminescent bacteria produce significant signal from the lungs (Figure 1). Luminescence intensity is quantified as total flux within a R.O.I (region of interested) (Figure 2). The quantitative data for light intensity is normalized for colony forming units (CFU) of bacteria obtained from lungs to confirm that the signal is produced from the bioluminescent bacteria and can be compared to negative control. The location and intensity of signal can be further analyzed by DLIT 3D reconstruction of the luminescence source based on tomography of the mouse surface11. These analyses allow quantification and localization of the bioluminescent signal produced. The results of 3D reconstruction of a luminescent source in infected mice demonstrates that light is produced from lungs of the mouse (Figure 3). The ex vivo images of the resulting mouse lungs confirm that luminescence is emitted from the lungs, rather than some other closely juxtaposed tissue or organ (Figure 2C).
<img src="/files/ftp_upload/2547/2547fig1.jpg" alt="Figure 1" /> 
Figure 1. Luminescence imaging of pulmonary infected mice with bioluminescent bacteria tagged with CBRluc. Uninfected control mouse is on the left and two infected mice are on the right. Mice were infected with bacteria expressing CBRluc (n=2) via the intratracheal route. 10 minutes after luciferin injection, luminescence images were acquired for 10 min at 4 positions: dorsal, ventral, left side and right side. 
<img src="/files/ftp_upload/2547/2547fig2.jpg" alt="Figure 2" /> 
Figure 2. The quantitative light intensity from mice infected with bacteria expressing CBRluc. (A, B) Luminescence images of ROI analysis and the quantified total flux of light from R.O.I. in dorsal and ventral positions, respectively. Uninfected mice are on the left and two infected mice are on the right. Luminescence images were acquired from lungs of mice uninfected and infected with CBRlux (n=2). The light intensity around lungs was quantified by ROI analysis. C) ex vivo images of lungs from uninfected (top) and infected (bottom two sets of lungs) mice. Luciferin was injected 5 miniuts before of euthanasia and images were then acquired. The quantified values are normalized to CFU.
<img src="/files/ftp_upload/2547/2547fig3.jpg" alt="Figure 3" /> 
Figure 3. The 3D reconstruction of bioluminescence source(s) from a pulmonary infected mouse. The mouse was infected with bacteria expressing CBRluc by intratracheal injection. The image sequence was acquired using different emission filters of serial wavelengths from 540 nm to 700 nm. An image sequence was used for 3D reconstitution for luminescence source in animal subjects that contained a structured imag. A) The tomography for mouse is shown in different positions: front, back, left and right. Light sources are shown as voxels (red boxes within 3D mouse) that are located within lung as determined by reconstruction. B) Photon densiy map of measured and simulated data. Comparing the measured and simulated photon density curves provide the information of quality of reconstruction. Good quality reconstructions result in similar measured and simulated photon densities.

<ol>
	<li>Wilson, T., Hastings, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence.">Bioluminescence.</a> Annu Rev Cell Dev Biol. 14, 197-230 (1998).</li><li>Contag, C. H., Bachmann, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+in+vivo+bioluminescence+imaging+of+gene+expression.">Advances in in vivo bioluminescence imaging of gene expression.</a> Annu Rev Biomed Eng. 4, 235-260 (2002).</li><li>Hutchens, M., Luker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+bioluminescence+imaging+to+the+study+of+infectious+diseases.">Applications of bioluminescence imaging to the study of infectious diseases.</a> Cell Microbiol. 9, 2315-2322 (2007).</li><li>Doyle, T. C., Burns, S. M., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+integrated+studies+of+infection.">In vivo bioluminescence imaging for integrated studies of infection.</a> Cell Microbiol. 6, 303-317 (2004).</li><li>Wood, K. V., Lam, Y. A., Seliger, H. H., McElroy, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+DNA+coding+click+beetle+luciferases+can+elicit+bioluminescence+of+different+colors.">Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors.</a> Science. 244, 700-702 (1989).</li><li>Wet, J. R. d. e., Wood, K. V., Helinski, D. R., DeLuca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+firefly+luciferase+cDNA+and+the+expression+of+active+luciferase+in+Escherichia+coli.">Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.</a> Proc Natl Acad Sci U S A. 82, 7870-7873 (1985).</li><li>Hastings, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistries+and+colors+of+bioluminescent+reactions:+a+review.">Chemistries and colors of bioluminescent reactions: a review.</a> Gene. 173, 5-11 (1996).</li><li>Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emission+spectra+of+bioluminescent+reporters+and+interaction+with+mammalian+tissue+determine+the+sensitivity+of+detection+in+vivo.">Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo.</a> J Biomed Opt. 10, 41210-41210 (2005).</li><li>Rice, B. W., Cable, M. D., Nelson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+light-emitting+probes.">In vivo imaging of light-emitting probes.</a> J Biomed Opt. 6, 432-440 (2001).</li><li>Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18, 593-603 (1995).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J Biomed Opt. 12, 024007-024007 (2007).</li><li>Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clearer+vision+for+in+vivo+imaging.">A clearer vision for in vivo imaging.</a> Nat Biotechnol. 19, 316-317 (2001).</li></ol>

Animal studies were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Commitee of Texas A&amp;M University.

Although following these protocols will usually result in high quality images, it is important to consider a few key issues in order to obtain accurate and consistent data from imaging studies. Luminescence images should be acquired that have counts from 600 to 60,000 to ensure that the signal is above background and the camera is not saturated. If signal obtained is less than 600 the exposure conditions should be adjusted to increase counts. If signal obtained is over 60,000 the camera is saturated in some regions. Wh...

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		<th>Comment</th>
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<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>VETONE</td>
<td>501027 </td>
<td>&nbsp;</td>
<tr>
<td>Ketamine </td>
<td>&nbsp;</td>
<td>Butler animal health supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine </td>
<td>&nbsp;</td>
<td>MP Biomedical</td>
<td>158307</td>
<td>&nbsp;</td>
<tr>
<td>Luciferin</td>
<td>&nbsp;</td>
<td>GMT</td>
<td>LUCK-100</td>
<td>&nbsp;</td>
<tr>
<td>Fetal plus solution</td>
<td>&nbsp;</td>
<td>VOR tech pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cathether (22G x 1&#x201D;)</td>
<td>&nbsp;</td>
<td>TERUMO</td>
<td>OX2225CA</td>
<td>&nbsp;</td>
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<td>Guide wire </td>
<td>&nbsp;</td>
<td>Hallowell EMC</td>
<td>210A3491</td>
<td>&nbsp;</td>
<tr>
<td>Octocope with speculum </td>
<td>&nbsp;</td>
<td>Hallowell EMC</td>
<td>000A3748</td>
<td>&nbsp;</td>
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<td>Xenogen IVIS system</td>
<td>&nbsp;</td>
<td>Caliper Life Sciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>XGI-8-gas Anesthsia System</td>
<td>&nbsp;</td>
<td>Caliper Life Sciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Living Imaging Software</td>
<td>&nbsp;</td>
<td>Caliper Life Sciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Transparent nose cones</td>
<td>&nbsp;</td>
<td>Caliper Life Sciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Light baffle divider </td>
<td>&nbsp;</td>
<td>Caliper Life Sciences</td>
<td>&nbsp;</td>
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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.

Watch this Scientific Journal Video about Using Luciferase to Image Bacterial Infections in Mice at JoVE.com

Using Luciferase to Image Bacterial Infections in Mice

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 ...

using-luciferase-to-image-bacterial-infections-in-mice

Research

JoVE Journal

Immunology and Infection

20.3K Views.  Texas A&M Health Science Center. 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.

Video: Using Luciferase to Image Bacterial Infections in Mice

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...