This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (Z01 ES102005).

All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review by the Animal Care and Use Committee of the NIEHS.
1. Preparation of K. pneumoniae Culture
Caution: Perform all steps in a biosafety level 2 (BSL2) hood or other BSL2 designated area and discard waste per institute BSL2 guidelines.
<ol>
	<li>For suspension growth of K. pneumoniae, thaw a glycerol stock of bacteria and inoculate a working culture by transferring 1 ml of K. pneumoniae stock to 50 ml of tryptic soy broth (TSB) in a 500 ml or larger flask.</li>
	<li>Make glycerol stocks by adding 900 &#181;l of cultured log-phase K. pneumoniae to 100 &#181;l of sterile glycerol and freezing at -20 &#176;C or -80 &#176;C. For long term storage, -80 &#176;C is recommended.</li>
	<li>Culture bacteria from step 1.1 by shaking flask overnight (14 - 18 hr) at 37 &#176;C at 200-225 rpm.In order to promote log phase growth, dilute 1-5 ml of this overnight culture into a flask containing 50 ml TSB in the morning and place back at 37 &#176;C with shaking for ~2.5 hr.</li>
	<li>Transfer 1 ml of K. pneumoniae culture from the last step into a 1.5 ml tube and spin at 7,500 x g for 2 min. A white pellet should be visible. Remove supernatant without disturbing the pellet, gently resuspend bacteria with pipette in 1 ml of sterile saline, and spin again at 7,500 x g for 2 min.
	<ol>
		<li>Perform this wash step 2x, bringing up the final, washed pellet in 1 ml of sterile saline.</li>
	</ol>
	</li>
	<li>Perform log-wise serial dilutions of this neat inoculum. For example, add 400 &#181;l of the inoculum into 3.6 ml of sterile saline serially to make a 10-1-10-9 dilution series.</li>
	<li>Determine the OD600 of washed K. pneumoniae by placing 1 ml of the 1:10 and 1:100 dilutions into disposable cuvettes and measuring the OD600, blanking first with sterile saline. Measure duplicates to ensure accuracy. An OD600 of 1.0 is roughly equivalent to 4-7 x 108 CFU/ml. Note that the relationship between OD and CFU/ml may not be linear.
	<ol>
		<li>Use the OD600 from the dilutions to calculate concentration of K. pneumoniae, applying the OD:CFU/ml conversion above, and factoring in the dilution.</li>
	</ol>
	</li>
	<li>Use the K. pneumoniae concentration to prepare the desired inoculum CFU dose in a &#60;100 &#181;l volume for delivery to mice (see below).</li>
	<li>Also plate 100 &#181;l of 10-6-10-9 dilutions and 100 &#181;l of sterile saline onto individual tryptic soy agar (TSA) plates at RT. Incubate at 37 &#176;C overnight and enumerate bacterial colonies in order to calculate exact concentration that was used in experiment and to control for contamination. 
	NOTE: Actual bacterial concentration of final inoculum may be &#177; 500 CFU/ml that predicted by absorbance. Experimenters should ideally confirm the OD600:CFU/ml relationship in pilot studies prior to use in vivo in mice, empirically readjusting the OD600:CFU/ml conversion based on their personal experience.</li>
</ol>
2. Murine Intratracheal (i.t.) Aspiration of K. pneumoniae from Oropharynx
<ol>
	<li>Ensure that mice are uniquely identified by tail mark, tattoo, or other approved identifier.</li>
	<li>Draw up dosing inoculum of bacteria using P200 pipette prior to removing mice from isoflurane in order to expedite procedure. For best results with i.t. aspiration, use a volume of &#60;100 &#181;l to prevent overflow. Herein, use 2000 CFU/50 &#181;l of K. pneumoniae.</li>
	<li>Anesthetize mice in a clear chamber with isoflurane gas (e.g., 2% isoflurane at flow rate of 1 L/min) or as per institutional guidelines. The number of mice to anesthetize together is determined by comfort level of the experimenter, typically 1-2&#160;at a time. Observe breathing and confirm level of anesthesia once deep breaths are visible and 2-3 sec can be counted between breaths. 
	NOTE: If there is concern about possible confounding effects of volatile (inhaled) anesthetics, anesthesia can be achieved instead with intraperitoneal injection of ketamine/xylazine. With the method described above, deep anesthesia only lasts a few min. If more prolonged anesthesia is induced, eye ointment should be used to prevent ocular desiccation.</li>
	<li>Once mouse is anesthetized, position it in a semirecumbent supine position, suspended by the maxillary incisors from a rubber band stretched between pegs on a slanted Plexiglas board (Figure 1).</li>
	<li>Gently pull the mouse tongue to the side with a pair of blunt non-ridged forceps and deposit dose into the oral cavity with a P200 pipette. Exercise great care to avoid inducing trauma to either the tongue or the oropharynx.</li>
	<li>While keeping the tongue retracted, gently occlude the nose with a gloved finger until the mouse inhales. Continue to cover the nose until the mouse has taken two or more inhalations, and no liquid is visible in the oral cavity. Covering the nose helps ensure that the mouse will inhale the bacteria into the lungs, as mice are obligate nose breathers.</li>
	<li>Remove the mouse from the inoculation board and return to its cage, placing the mouse on its back to prevent bedding or debris from blocking the nares while the mouse is recovering from anesthesia.</li>
	<li>Once all mice have been dosed and have awoken from anesthesia, house mice in a cubicle/room containing only mice that have been dosed with K. pneumoniae. Monitor mice daily, including body weights if going beyond 48 hr post-infection.</li>
	<li>Use daily mash and/or supplemental heat if allowing the mice to go beyond 48 hr.</li>
</ol>
3. Bronchoalveolar Lavage Fluid (BALF) Collection and Analysis
<ol>
	<li>Euthanize mice per institutional guidelines. Here, use a lethal injection of 150 mg/kg sodium&#160;pentobarbital &#160;or equivalent dose of commercial euthanasia solution&#160;followed by exsanguination, as this avoids possible complications to the lungs associated with CO2 inhalation and cervical dislocation.</li>
	<li>Position mouse on back and sterilize mouse by spraying fur with 70% ethanol particularly over the chest and neck area.</li>
	<li>Make a longitudinal cut just below the sternum, then holding the sternum with forceps, nick the diaphragm, allowing the lung to fall back into the chest cavity. Cut up through the chest cavity on either side of the vasculature and then up through the neck, exposing the trachea.</li>
	<li>As the trachea is surrounded by longitudinal muscles on either side, carefully cut through the middle and push tissue to the sides, as this avoids cutting the surrounding vasculature, which could introduce blood into the trachea. If the vasculature is nicked, clean the surrounding area with gauze prior to accessing the tracheal lumen.</li>
	<li>Use surgical scissors or a needle to nick the trachea about &#188; of the way down from the head and insert a cannula attached to a 1 ml syringe preloaded with 1 ml of 1x phosphate-buffered saline (PBS) caudally into the trachea. If using mice &#60;8 weeks of age or females, reduce the lavaging volume to 600-800 &#181;l.</li>
	<li>Push the volume into the lung slowly, allowing all lobes to inflate and then pull the volume back out with the syringe, repeating 3x. If PBS is coming out of the nostrils the cannula has not been pushed far enough into the trachea or inflation is occurring too quickly. Alternatively, if the lungs are not inflating well, the cannula has been pushed in too far, so withdraw slightly.</li>
	<li>Collect pooled washes in a 15 ml tube on ice.</li>
	<li>Spin the airspace lavage fluid at 1,200 x g for 5 min, collect supernatant into a 1.5 ml tube and freeze at -80 &#176;C. Subsequently, use the supernatant (i.e., BALF) for measurement of protein, cytokines, and for various other biochemical assays. The pellet represents cells from the bronchoalveolar space.</li>
	<li>If red blood cells (RBCs) are evident in the cell pellet based on color, lyse with 1 ml of ACK lysis buffer for 1 min followed by addition of 5 ml of 1x PBS to stop the lysis reaction and dilute the buffer. Handle all specimens identically, either treated or not with ACK lysis buffer.</li>
	<li>Spin cells at 1,200 x g for 5 min, decant the supernatant, and bring the cells up in 1 ml of 1x PBS.</li>
	<li>Vortex and count cells on a hemocytometer for enumeration of total airspace cells.</li>
	<li>Based on density of cells add approximately 80-150 &#181;l (~80 &#181;l for an infected mouse and ~150 for a naive mouse) to a slide chamber on a cytospin centrifuge. Spin cells onto microscope slides for subsequent staining to determine cell differential populations.</li>
	<li>Allow cells to dry on slides overnight. Stain cells with tri-stain (e.g., Hema 3 [see table]) by dipping the slides 7 times in fixative, 9 times in stain 1, and 7 times in stain 2 (no rinses between stains), and allow to dry. After stain has dried, coverslip slides and manually count differentials by microscope. Generally, neutrophils and monocyte/macrophages are easily distinguished by their size and nuclear morphology.</li>
</ol>
4. Determining Bacterial Load in Lung and Peripheral Tissues
<ol>
	<li>Before beginning: prepare dilution tubes with 900 &#181;l of 1x PBS for lung and spleen and 90 &#181;l of 1x PBS for blood. Label plates for culturing bacteria and set at room temperature; therefore, once tissue has been collected time will be minimized between homogenization and plating for bacterial growth. 
	Note: Due to the heterogeneous deposition of bacteria in the lungs that can occur with aspiration, it is recommended that individual mice be used for analysis of either total airway cellularity or total (bilateral) lung bacterial burden.</li>
	<li>Euthanize the mouse as per step 3.1 and subsequently collect blood from the left ventricle, placing into a heparinized tube to avoid clotting. Remove all lung lobes and place in a 15 ml tube containing 5 ml of 1x PBS, followed by removal of the spleen and placement into 2 ml of 1x PBS. Place tubes on ice. Take care to clean instruments with ethanol between each tissue/mouse.</li>
	<li>Homogenize the tissue on ice, preferably with disposable homogenizers that can be changed between each sample. Disposable homogenizer tips can be autoclaved for reuse between experiments.</li>
	<li>Once tissue has been homogenized, perform serial dilutions and plate. Plate samples of a given tissue/dilution in duplicate on a single TSA plate by drawing a dividing line on the plastic. Plating and dilution suggestions are shown below (in all cases, 10 &#181;l of sample is spread onto plate) and are based on a 24 hr post-infection necropsy. If analyzing 48-72 hr samples, a larger number of dilutions may need to be plated due to increased bacterial burden at later time points.
	<ol>
		<li>For Blood - dilute 1:10 by adding 10 &#181;l of blood into 90 &#181;l of sterile 1x PBS. Plate neat and 1:10 diluted samples in duplicate. Once blood has been plated, spin residual blood at 9,300 x g for 10 min to extract plasma for measurement of cytokines and chemokines.</li>
		<li>For Lung - dilute 1:10 - 1:100 by adding 100 &#181;l of homogenized lung into 900 &#181;l of sterile 1x PBS serially. Plate neat homogenate and all dilutions in duplicate.</li>
		<li>For Spleen - dilute 1:10-1:100 by adding 100 &#181;l of homogenized spleen into 900 &#181;l of sterile 1x PBS serially. Plate neat homogenate and both dilutions in duplicate.</li>
	</ol>
	</li>
	<li>Allow spread samples to dry briefly.</li>
	<li>Invert TSA plates and place in a static 37 &#176;C incubator overnight.</li>
	<li>Determine the number of colonizing bacteria by averaging the bacterial colonies from both sides of the plate and from each dilution. Factor in the initial dilutions of 5 ml for lung and 2 ml for spleen to determine total tissue CFUs.</li>
</ol>

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The authors declare that they have no competing financial interests.

Murine models of bacterial pneumonia, partnered with gene targeting and in vivo biological and pharmacological interventions, have provided critical insights into the pulmonary host defense response. Great advances have been made in particular in our understanding of the chemokines and adhesion molecules that govern recruitment of neutrophils to the infected airspace10,11. In vivo models of pneumonia, unlike cell-based or alternative approaches, have also provided key insights into endocrine ...

Community-acquired pneumonia remains the leading cause of death from infection in the U.S., with little overall change in mortality rates over the past 40 years despite improvements in vaccination and antibiotic strategies1,2. Despite the lack of perceptible progress at the public health level, in recent years dramatic advances have been made in our understanding of the molecular and cellular pathogenesis of pneumonia, with many of these steps forward made possible by the use of mouse models of lung infection. The genetic tractability of the mouse, the similarity of the murine and human immune systems, and the vast array of murine-targeted immunologic reagents that have become commercially available have together facilitated rapid progress of the field.
Mouse models of bacterial pneumonia described in the literature have generally relied upon one of four technical routes for pathogen inoculation: i) aerosolization; ii) intranasal delivery; iii) peroral delivery; and iv) surgical intratracheal injection (i.e., tracheotomy)3. All routes of infection have advantages and disadvantages3. In particular, relative exposure of the upper airway, potential for admixture of oronasal flora, requirements for general anesthesia, variability of the inoculum delivered to the distal lung, lobar distribution of the delivered pathogens, technical expertise requirements, and procedural morbidity vary widely across these approaches.
Commonly used peroral infection techniques include endotracheal (translaryngeal) cannulation via either a &#39;blind&#39; (non-visualized) approach, or under direct laryngeal visualization3-5. Both methods, while robust, require substantial training and also carry risk of trauma to the upper airway. In the present report, we describe a technically non-intensive, non-invasive, inexpensive, and rapid method of peroral infection, whereby bacteria (Klebsiella pneumoniae in the example provided) pipetted into the oropharynx of an anesthetized mouse are delivered to the lungs via aspiration (i.e., inhalation). We and others have used the aspiration pneumonia technique successfully6-9. This versatile and easily learned lung-delivery method can be extended to the delivery of many additional non-caustic agents to the lungs, including cytokines and other proteins, pathogen-associated molecules (e.g., lipopolysaccharide), cells (i.e., adoptive transfer), and toxins (e.g., bleomycin). In addition to discussing important technical considerations, we also describe an integrated, quantitative approach for assessing the subsequent host response to pneumonia, including downstream measurement of bacterial clearance (i.e., colony forming unit [CFU] quantitation in the lung and peripheral organs) and leukocyte accumulation in the airspace.

<table><tbody><tr><td>Klebsiella pneumoniae, serotype 2</td><td>ATCC</td><td>43816</td><td></td></tr><tr><td>Tryptic soy broth</td><td>Becton Dickenson</td><td>211825</td><td></td></tr><tr><td>Excel Safelet IV Catheters, 18 G x 1 1/4&quot;</td><td>Claflin Medical Equipment</td><td>MEDC-031122</td><td></td></tr><tr><td>Hema 3 Solution 1</td><td>Fisher</td><td>23-122-937</td><td></td></tr><tr><td>Hema 3 Solution 2</td><td>Fisher</td><td>23-122-952</td><td></td></tr><tr><td>Hema 3 Fixative</td><td>Fisher</td><td>23-122-929</td><td></td></tr><tr><td>27&amp;frac12; gauge tuberculin syringes</td><td>Fisher</td><td>14-826-87</td><td></td></tr><tr><td>Lithium heparin plasma collectors</td><td>Fisher</td><td>2675187</td><td></td></tr><tr><td>L-shaped disposable spreaders</td><td>Lab Scientific</td><td>DSC</td><td></td></tr><tr><td>1x PBS, pH 7.4</td><td>prepared in-house</td><td>n/a</td><td>Distilled water (5 L), NaCl (40 g), KCl (1 g), Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (5.75 g), KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (1 g)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>ACK lysis buffer</td><td>prepared in-house</td><td>n/a</td><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl (4.145 g), KHCO&lt;sub&gt;3&lt;/sub&gt; (0.5 g), EDTA (18.6 mg), bring up to 500 ml with distilled water and pH to 7.4</td></tr></tbody></table>

a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

C57BL/6 mice were infected with 2000 CFU of K. pneumoniae 43816 (serotype 2) via oropharyngeal aspiration into the lungs. At this dose, mice typically begin to show clinical symptoms 12-24 hr post-infection including lethargy, ruffled fur, and weight loss of 5-10% (Figure 2A). Within 48-72 hr post-infection, many of the mice show symptoms of illness and morbidity that is typically preceded by an average of 20% weight loss and results in hunched postures with decr...

Watch this Scientific Journal Video about A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice at JoVE.com

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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Immunology and Infection

8.9K Views.  National Institutes of Health.  Verscheidene werkwijzen zijn beschreven in de literatuur voor het modelleren van bacteriële pneumonie bij muizen. Hierin beschrijven we een niet-invasieve, goedkope, snelle werkwijze voor het induceren via aspiratie pneumonie (bijvoorbeeld inhalatie) van een bacterieel inoculum gepipetteerd in de orofarynx. Downstream methoden voor de beoordeling van de pulmonaire aangeboren immuunrespons zijn ook gedetailleerd. 

Video: A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

Medicine

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

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