This work was supported by U.S. Public Health Service grant AI103806 from the National Institutes of Health.

All animals are to be maintained and handled with maximum care to minimize discomfort during the course of the procedure. The procedure is to be conducted in compliance with the Institutional Animal Care and Use Committee and all federal, state and local laws. Also note that the laboratory experiments are to be conducted in accordance with Biosafety Level 2 guidelines.
1. Growth of L. monocytogenes for Mouse Infection Studies
<ol>
	<li>Autoclave 500 mL of Brain Heart Infusion (BHI) agar medium in a 1 L Erlenmeyer flask to prepare solid media plates. Allow the media to cool in a 56 &#176;C water bath and then add streptomycin at a final concentration of 100 &#181;g/mL.
	<ol>
		<li>Pour 25 mL of media/Petri dish into Petri dish plates. Allow the plates to dry at room temperature (22 oC-25 &#176;C). If needed, the plates can be dried at 37 &#176;C until completely dry.</li>
	</ol>
	</li>
	<li>Using a sterile inoculating loop and aseptic technique, obtain a loop full of frozen L. monocytogenes strain 10403S11,12 from a stock culture frozen in BHI media plus 30% glycerol and maintain in a -80 &#176;C freezer.
	<ol>
		<li>Streak the L. monocytogenes on a BHI agar/streptomycin plate such that single bacterial colonies can be isolated. Place the plates in a 37 &#176;C incubator overnight (18 h to 24 h) to grow the L. monocytogenes colonies.</li>
		<li>Using a sterile inoculating loop, pick a single L. monocytogenes colony from the BHI agar plate and inoculate the colony into a sterile test tube containing 5 mL of BHI broth containing 100 &#181;g/mL streptomycin.</li>
	</ol>
	</li>
	<li>Incubate the L. monocytogenes culture tube slightly tilted at 30&#176; overnight in a static incubator.
	<ol>
		<li>The following day, visually inspect the L. monocytogenes culture for growth (the BHI media will be turbid) and vortex briefly to ensure a uniform suspension of the culture.</li>
		<li>Aseptically remove a 1 mL aliquot of the bacterial culture and measure the optical density at 600 nm (OD600) using a spectrophotometer. 
		NOTE: Sterile BHI broth is used as the blank for the spectrophotometer reading before measuring the optical density of the L. monocytogenes culture. The L. monocytogenes culture can also be diluted (two to five-fold) in BHI before reading the optical density.</li>
		<li>Determine the number of L. monocytogenes colony forming units (CFU) per mL of BHI culture by plating 10-fold serial dilutions of the culture. This is useful to establish a relationship between the optical density of the L. monocytogenes culture and the number of bacteria per mL of culture. In general, an OD600 of 1.0 for a L. monocytogenes culture equals approximately 1 x 109 CFU of bacteria per mL.</li>
	</ol>
	</li>
</ol>
2. Preparation of L. monocytogenes for Infection of Mice
<ol>
	<li>Eighteen to twenty-four hours prior to animal infection, grow L. monocytogenes cultures in BHI broth containing 100 &#181;g/mL streptomycin and determine the OD600/~CFU per mL as indicated in step 1. 
	NOTE: Mice are generally ordered one week in advance of infection and are acclimated to the animal facility before infection studies are performed. In this study, 6&#8211;8 weeks old female BALB/c mice (5 mice&#160;per group) were housed in a barrier environment in the Harvard Medical School BSL2-level animal containment facility and supplied food and water ad libitum.</li>
	<li>Determine the amount of L. monocytogenes culture required to infect each mouse based on the ~CFU per mL of bacterial culture. Make a dilution of the L. monocytogenes culture in sterile phosphate-buffered saline pH 7.2 (PBS) to the appropriate concentration of bacteria. Mice are typically infected intravenously with 200 &#181;L of PBS containing 1&#8211;2 &#215; 104 CFU of bacteria/animal.</li>
	<li>Verify the number of L. monocytogenes in the inoculum by plating 10-fold serial dilutions onto BHI agar plates containing 100 &#181;g/mL streptomycin. Incubate the plates in a 37 &#176;C incubator overnight to determine the number of L. monocytogenes inoculated per mouse as follows: 
	Inoculum (CFU) = (number of colonies x dilution factor) / mL plated x volume (mL) injected</li>
</ol>
3. Infection of Mice with L. monocytogenes via Intravenous Tail Vein Injection
<ol>
	<li>Remove the lid and food bins from the cage containing the mice, and place the cage under a 250 W infrared heat lamp for 5 min. 
	NOTE: This method allows the tail veins to dilate, ensuring that injections are easier to perform.
	<ol>
		<li>Carefully place the animal in an appropriately sized mouse restraining device to safely restrain the animal during tail vein injections.</li>
	</ol>
	</li>
	<li>Mix the L. monocytogenes inoculum (step 2.2) and load the inoculum into a 1 mL syringe equipped with a 26 G needle.</li>
	<li>Locate a lateral tail vein of the mouse and clean the injection site using an alcohol pad or a 75% ethanol spray.</li>
	<li>Gently inject the mouse with 200 &#181;L of the L. monocytogenes suspension into a lateral tail vein using the syringe with the 26 G needle.
	<ol>
		<li>Use cotton gauze to briefly apply pressure on the injection site to stop any bleeding, and place the animal in a new cage. 
		NOTE: Perform this process for all of the mice to be injected and mark the cage with appropriate labels. To determine the post-injection concentration of the L. monocytogenes inoculum, repeat step 2.3 using the remaining amount of inoculum. The intravenous L. monocytogenes infection inoculum per mouse is reported as the average CFU measured from the pre- and post-injection inoculum samples.</li>
	</ol>
	</li>
	<li>Monitor the infected animals daily and note any overt signs of illness (e.g., ruffled fur, hunched posture, sluggish movement, weight loss). Remove animals that appear to be significantly moribund prior to 72 h post-infection (e.g., 24, 48 h post-infection) from the study and humanely sacrifice. Continue daily monitoring until all remaining animals are sacrificed at 72 h post-infection. 
	NOTE: The 50% lethal dose (LD50) for L. monocytogenes strain 10403S in BALB/c mice is ~1&#8211;2 x 104 CFU/animal. Mice infected with LD50 doses of L. monocytogenes using this protocol may exhibit signs of illness, as indicated above, and investigators could expect the mice to start succumbing to the infection following 72 h post-infection.</li>
</ol>
4. Dissection and Cardiac Perfusion of Mice Infected with L. monocytogenes 
<ol>
	<li>At 72 h post-infection (or at an earlier desired time point), euthanize the infected mice using a protocol approved by the institutional Animal Ethics Committee, such as CO2 asphyxiation followed by cervical dislocation. 
	NOTE: If blood collection is to be performed, minimize tearing of the blood vessels while performing cervical dislocation.
	<ol>
		<li>Confirm mice have been euthanized by the absence of a paw-twitch response. 
		NOTE: If cardiac perfusion is to be performed, set up an automated pump/perfusion system at a flow rate of 4 mL volume/min.</li>
	</ol>
	</li>
	<li>Place the animal on its back in a dissection pan and apply 75% ethanol on the abdominal fur/skin surface.</li>
	<li>Using scissors, make an incision in the abdominal wall and expand the incision to just below the rib cage.</li>
	<li>Expose the diaphragm and visceral organs. 
	NOTE: Be careful not to lacerate any visceral organs.</li>
	<li>Open the thoracic cavity by cutting the diaphragm and cut the rib cage bilaterally to expose the left ventricle of the heart.</li>
	<li>Using blunt forceps, gently grasp the heart and insert a 21 G butterfly needle connected to a perfusion system into the left ventricle and then carefully cut the right atrium to collect the blood (~0.2 mL) into a 2 mL tube containing 10 mM ethylenediaminetetraacetic acid (EDTA) to prevent coagulation. A schematic diagram of cardiac perfusion in the mouse is shown in Figure 2. 
	NOTE: If perfusion is not to be performed, blood may be collected by cardiac puncture using a 1 mL syringe and quickly transferred into a 2 mL tube containing 10 mM EDTA to prevent coagulation. L. monocytogenes-infected organs are then harvested as described (step 5.1).</li>
	<li>Begin perfusion and perfuse the animal for 4 min with 15&#8211;20 mL of PBS containing 10 mM EDTA. 
	NOTE: Assess perfusion by observing the blood and PBS-EDTA solution flowing through the right atrium and into the dissection pan. The brain and liver will appear blanched after complete perfusion ensuring that remaining bacteria are from the harvested organs and not the circulating blood.</li>
</ol>
5. Organ Harvesting and Determination of Bacterial Burdens
<ol>
	<li>Following perfusion, harvest organs (e.g., liver, spleen) by carefully removing any vasculature and ligaments attached and place the organs separately into a sterile 15 mL conical tube containing 5 mL of sterile cold (4 &#176;C) PBS. Store samples on ice until further processing occurs.</li>
	<li>To harvest the brain, decapitate the head behind the ears using scissors and cut the scalp skin in-between the animal&#39;s eyes down the midline. If required, trim excess tissue keeping the scissors pressed against the skull.
	<ol>
		<li>Gently insert one tip of the scissors into the foramen magnum (the hole in the base of the skull through which the spinal cord passes) and cut laterally into the skull on both sides.</li>
		<li>Gently cut the skull in-between the rodent&#39;s eyes down the midline, being sure to apply lateral pressure. Avoid perturbation of the brain and maintain minimum contact between the brain tissue and the scissors.</li>
		<li>Using fine tip forceps, open the skull to expose the brain and place a spatula between the underside of the brain and the base of the skull to remove the brain. 
		NOTE: This results in tearing of brain nerve fibers.</li>
		<li>Carefully transfer the brain to a 15 mL conical tube containing 5 mL of sterile cold PBS.</li>
		<li>Discard the mouse carcass, and repeat these steps for the remaining animals. 
		NOTE: Once all organs have been harvested, clean the procedure area and prepare for the organ homogenization to determine bacterial burdens.</li>
	</ol>
	</li>
	<li>Clean and sterilize the tip of a tissue homogenizer placed in a biosafety cabinet by inserting the tip and running the homogenizer for 10 s sequentially in 5% bleach, sterile water, 75% ethanol, sterile water each contained in a separate 15 mL conical tube.</li>
	<li>Homogenize the infected brain (~20 s on setting 6, maximum rpm) in the 15 mL conical tube until no visible tissue fragments remain.
	<ol>
		<li>Clean the homogenizer as described in step 5.3 to prevent bacterial carry over contamination to the next organ sample.</li>
		<li>Perform the homogenization process for all other organs (e.g., liver, spleen).</li>
		<li>Once the homogenization process is completed, clean the homogenizer as described in step 5.3 and store until further use.</li>
	</ol>
	</li>
	<li>Prepare 10-fold serial dilutions of the organ homogenates in sterile PBS and plate the dilutions on BHI agar/streptomycin plates to determine the CFU per organ. 
	NOTE: A similar method is performed to determine the CFU per mL of blood.
	<ol>
		<li>After all dilutions are plated, transfer the plates to&#160;a 37 &#176;C incubator overnight.</li>
		<li>The following day, count the number of colonies on each plate to determine the total number of bacteria per organ or mL of blood as follows: 
		CFU/organ = (number of colonies x dilution factor) / mL of homogenate plated x 5 
		CFU/mL of blood = (number of colonies x dilution factor) / mL of blood plated</li>
	</ol>
	</li>
</ol>

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	<li>Radoshevich, L., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes:+towards+a+complete+picture+of+its+physiology+and+pathogenesis.">Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis.</a> Nature Reviews Microbiology. 16 (1), 32-46 (2018).</li><li>de Noordhout, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+listeriosis:+a+systematic+review+and+meta-analysis.">The global burden of listeriosis: a systematic review and meta-analysis.</a> The Lancet Infectious Diseases. 14 (11), 1073-1082 (2014).</li><li>Drevets, D. A., Leenen, P. J., Greenfield, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+the+central+nervous+system+by+intracellular+bacteria.">Invasion of the central nervous system by intracellular bacteria.</a> Clinical Microbiology Reviews. 17 (2), 323-347 (2004).</li><li>Huang, S. H., Stins, M. F., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+penetration+across+the+blood-brain+barrier+during+the+development+of+neonatal+meningitis.">Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis.</a> Microbes and Infection. 2 (10), 1237-1244 (2000).</li><li>Brouwer, M. C., Tunkel, A. R., van de Beek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+diagnosis,+and+antimicrobial+treatment+of+acute+bacterial+meningitis.">Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis.</a> Clinical Microbiology Reviews. 23 (3), 467-492 (2010).</li><li>Thigpen, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+meningitis+in+the+United+States,+1998-2007.">Bacterial meningitis in the United States, 1998-2007.</a> New England Journal of Medicine. 364 (21), 2016-2025 (2011).</li><li>Durand, M. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ROCK+activity+allows+InlF-mediated+invasion+and+increased+virulence+of+Listeria+monocytogenes.">Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.</a> Molecular Microbiology. 68 (3), 749-767 (2008).</li><li>Grubaugh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VirAB+ABC+transporter+is+required+for+VirR+regulation+of+Listeria+monocytogenes+virulence+and+resistance+to+nisin.">The VirAB ABC transporter is required for VirR regulation of Listeria monocytogenes virulence and resistance to nisin.</a> Infection and Immunity. 86 (3), 901-917 (2018).</li><li>Vazquez-Boland, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+pathogenesis+and+molecular+virulence+determinants.">Listeria pathogenesis and molecular virulence determinants.</a> Clinical Microbiology Reviews. 14 (3), 584-640 (2001).</li><li>Ghosh, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+the+brain+by+Listeria+monocytogenes+is+mediated+by+InlF+and+host+cell+vimentin.">Invasion of the brain by Listeria monocytogenes is mediated by InlF and host cell vimentin.</a> MBio. 9 (1), e00160-e00118 (2018).</li><li>Henke, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+spreads+within+the+brain+by+actin-based+intra-axonal+migration.">Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration.</a> Infection and Immunity. 83 (6), 2409-2419 (2015).</li><li>Maury, M. 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The authors declare no competing financial interests.

L. monocytogenes is able to cause life-threatening meningoencephalitis in humans. Prior studies have demonstrated the ability of bacteria to cross the blood-brain-barrier (BBB) and to colonize the brain. Three routes of brain invasion have been proposed during infection: direct penetration of the BBB by bacteria, stealth transport by bacteria contained inside of mononuclear cells3, and axonal migration by L. monocytogenes strains that cause rhombencephalitis15</...

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen and one of the most deadly food-borne pathogens worldwide. Ingestion of L. monocytogenes contaminated food can lead to listeriosis in humans, a severe invasive disease targeting mostly pregnant women, newborns, the elderly, and immunocompromised individuals1. L. monocytogenes is among the leading causes of death by a food-borne pathogen in the U.S. and case fatality rates from listeriosis are as high as 20&#8211;30%, the highest for all food-borne pathogens2. No vaccine currently exists for L. monocytogenes and the ability of bacteria to effectively spread to distal organs and the brain by crossing the blood-brain barrier (BBB) may lead to life-threatening meningitis and colonization of the brain3,4,5,6. Bacterial meningitis is typically severe; while most people who receive treatment recover, infections can cause serious complications, e.g., brain damage, hearing loss, or learning disabilities in children. L. monocytogenes is predicted to account for at least 10% of all community acquired meningitis in the U.S.7.
A major route for bacterial dissemination to the brain and meninges is through the bloodstream. Bacteria circulating in blood vessels in the brain are able to cross the BBB to cause brain infection. The BBB is a highly vascularized barrier system that protects the brain from foreign particles circulating in the blood. Endothelial cells constitute a layer that lines the interior surface of the blood vessels8,9. In addition to L. monocytogenes, multiple bacterial pathogens such as Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, and Haemophilus influenzae type b (Hib) are capable of colonizing the brain by crossing the BBB3,4,5,6. However, when examining bacterial burdens in the brain of infected mice, it is important to determine whether bacteria have crossed the BBB, otherwise the bacterial burden in the brain may represent bacteria that are still in the blood vessels of the brain. Thus, it is necessary to perfuse the mice of all blood prior to determining colony-forming units (CFU) of brain homogenates.
In this study, we describe in vivo methods to examine L. monocytogenes infection of the brain. For the methods described here, we used L. monocytogenes strain 10403S. L. monocytogenes&#160;10403S is one of the most widely used laboratory strains to study systemic listeriosis in the mouse model of infection10. This protocol is based on standard intravenous injection of L. monocytogenes followed by perfusion of the mice. A schematic outline of the infection protocol in mice is shown in Figure 1. L. monocytogenes-infected brains and other organs from non-perfused or perfused mice were collected and the bacterial organ burden determined. These methods are useful for not only determining total bacterial colonization of the brain in infected animals, but are also beneficial for determining whether bacteria have penetrated the BBB in vivo to mediate invasion of the brain. It is important to highlight that this laboratory protocol should be conducted following consultation with the relevant institutional biosafety committee and animal facility management.

<table><tbody><tr><td>Brain Heart Infusion media</td><td>Becton Dickinson</td><td>237200</td><td></td></tr><tr><td>Streptomycin sulfate&amp;nbsp;</td><td>Amresco</td><td>0382-50G</td><td></td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>Glycerol</td><td>VWR</td><td>97062-832</td><td></td></tr><tr><td>IKA T18 ULTRA-TURRAX Basic Homogenizer</td><td>IKA</td><td>3352109</td><td>Model: T18BS1</td></tr><tr><td>Spectrophotometer</td><td>Beckman Coulter</td><td>DU 800 series</td><td></td></tr><tr><td>BALB/c mice</td><td>Jackson Laboratory</td><td>Model #000651</td><td></td></tr><tr><td>1 mL syringes</td><td>Becton Dickinson</td><td>309659</td><td></td></tr><tr><td>26 G needles</td><td>Becton Dickinson</td><td>305115</td><td></td></tr><tr><td>21 G butterfly needles</td><td>Becton Dickinson</td><td>367281</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>60004</td><td></td></tr><tr><td>15 mL conical tubes</td><td>VWR</td><td>21008-918</td><td></td></tr><tr><td>Round-bottom test tubes</td><td>VWR</td><td>60819-546</td><td></td></tr><tr><td>Phosphate-buffered saline&amp;nbsp;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>Stainless steel spatula</td><td>VWR</td><td>82027-520</td><td></td></tr><tr><td>Stainless steel scissors (6.5 in)</td><td>VWR</td><td>82027-592</td><td></td></tr><tr><td>Stainless steel scissors (4.5 in)</td><td>VWR</td><td>82027-578</td><td></td></tr><tr><td>Stainless steel blunt forceps&amp;nbsp; (4.5 in)</td><td>VWR</td><td>82027-440</td><td></td></tr><tr><td>Stainless steel fine tip forceps&amp;nbsp; (6 in)</td><td>VWR</td><td>82027-406</td><td></td></tr></tbody></table>

listeria monocytogenes infection of the brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

The brain is highly vascularized and L. monocytogenes is known to infect cell types present in the blood3,13. The described protocol is used to demonstrate the ability of L. monocytogenes to cross the blood-brain barrier (BBB) leading to infection of the brain in mice. To determine if bacteria have crossed the BBB in vivo, perfusion of blood in the mouse is performed prior to determining bacterial burden...

Watch this Scientific Journal Video about Listeria monocytogenes Infection of the Brain at JoVE.com

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

listeria-monocytogenes-infection-of-the-brain

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Immunology and Infection

12.6K Views.  Harvard Medical School. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het bacteriële pathogenese veld over de vraag of door bloed overgedragen bacteriën de bloed-hersenbarrière kunnen passeren om het centrale zenuwstelsel en de hersenen te infecteren. Met deze techniek kunnen bacteriële getallen in het bloed en hersenweefsel van geïnfecteerde dieren worden bepaald, terwijl het aantal bacteriën dat zich in de bloedvaten van de hersenen bevat, uitsluit. Het aantonen van de procedure zal ...

Video: Listeria monocytogenes Infection of the Brain

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 ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

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 ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Source: Ghosh, P., et al., <a href="https://app.jove.com/v/58723">Listeria monocytogenes Infection of the Brain.</a> J. Vis. Exp. (2018)
This video demonstrates the quantification of the infective bacterial load from the perfused mouse brain infected with Listeria monocytogenes.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Organ Harvesting and ...

Listeria monocytogenes Infection of the Brain

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