The authors would like to thank Anna Walduck, Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan and Jonathan Wilksch for advice and reagents. This work was funded by the Juvenile Diabetes Research Foundation (1-2008-602) and the Australian National Health and Medical Research Council (575552). NW is supported by an Australian Postgraduate Award. OW is supported by a R.D. Wright Fellowship from the Australian National Health Medical Research Council.

<ol>
	<li>Wing, E. J., Gregory, S. H. <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:+clinical+and+experimental+update.">Listeria monocytogenes: clinical and experimental update.</a> J Infect Dis. 185, S18-S24 (2002).</li><li>Conlan, 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=Early+host-pathogen+interactions+in+the+liver+and+spleen+during+systemic+murine+listeriosis:+an+overview.">Early host-pathogen interactions in the liver and spleen during systemic murine listeriosis: an overview.</a> Immunobiology. 201, 178-187 (1999).</li><li>Neuenhahn, M., Busch, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+defenses+in+the+liver+during+Listeria+infection.">Innate defenses in the liver during Listeria infection.</a> Immunol Rev. 174, 150-159 (2000).</li><li>Hamon, M., Bierne, H., 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:+a+multifaceted+model.">Listeria monocytogenes: a multifaceted model.</a> Nat Rev Microbiol. 4, 423-434 (2006).</li><li>Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+Listeria+monocytogenes.">Immune responses to Listeria monocytogenes.</a> Nat Rev Immunol. 4, 812-823 (2004).</li><li>Cheers, C., McKenzie, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+and+susceptibility+of+mice+to+bacterial+infection:+genetics+of+listeriosis.">Resistance and susceptibility of mice to bacterial infection: genetics of listeriosis.</a> Infect Immun. 19, 755-762 (1978).</li><li>Garifulin, O., Boyartchuk, V. <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+as+a+probe+of+immune+function.">Listeria monocytogenes as a probe of immune function.</a> Brief Funct Genomic Proteomic. 4, 258-269 (2005).</li><li>Gervais, F., Stevenson, M., Skamene, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+resistance+to+Listeria+monocytogenes:+regulation+of+leukocyte+inflammatory+responses+by+the+Hc+locus.">Genetic control of resistance to Listeria monocytogenes: regulation of leukocyte inflammatory responses by the Hc locus.</a> J Immunol. 132, 2078-2083 (1984).</li><li>Gervais, F., Desforges, C., Skamene, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C5-sufficient+A/J+congenic+mouse+strain.+Inflammatory+response+and+resistance+to+Listeria+monocytogenes.">The C5-sufficient A/J congenic mouse strain. Inflammatory response and resistance to Listeria monocytogenes.</a> J Immunol. 142, 2057-2060 (1989).</li><li>Boyartchuk, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multigenic+control+of+Listeria+monocytogenes+susceptibility+in+mice.">Multigenic control of Listeria monocytogenes susceptibility in mice.</a> Nat Genet. 27, 259-260 (2001).</li><li>Boyartchuk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+resistance+locus+sst1+controls+innate+immunity+to+Listeria+monocytogenes+infection+in+immunodeficient+mice.">The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice.</a> J Immunol. 173, 5112-5120 (2004).</li><li>Pan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipr1+gene+mediates+innate+immunity+to+tuberculosis.">Ipr1 gene mediates innate immunity to tuberculosis.</a> Nature. 434, 767-772 (2005).</li><li>Garifulin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irf3+polymorphism+alters+induction+of+interferon+beta+in+response+to+Listeria+monocytogenes+infection.">Irf3 polymorphism alters induction of interferon beta in response to Listeria monocytogenes infection.</a> PLoS Genet. 3, 1587-1597 (2007).</li><li>Hof, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+different+strains+of+Listeria+monocytogenes+serovar+1/2a.">Virulence of different strains of Listeria monocytogenes serovar 1/2a.</a> Med Microbiol Immunol (Berl. 173, 207-218 (1984).</li><li>Wollert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+host+range+of+Listeria+monocytogenes+by+rational+protein+design.">Extending the host range of Listeria monocytogenes by rational protein design.</a> Cell. 129, 891-902 (2007).</li><li>Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+listeriosis:+role+of+internalin+in+crossing+the+intestinal+barrier.">A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.</a> Science. 292, 1722-1725 (2001).</li><li>Marco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+Listeria+monocytogenes+in+mice+infected+by+the+oral+route.">Penetration of Listeria monocytogenes in mice infected by the oral route.</a> Microb Pathog. 23, 255-263 (1997).</li><li>Busch, D. H., Vijh, S., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+for+infection+with+Listeria+monocytogenes.">Animal model for infection with Listeria monocytogenes.</a> Curr Protoc Immunol. Chapter. 19, 19-19 (2001).</li><li>Neuenhahn, M., Schiemann, M., Busch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+in+mouse+models+of+intracellular+bacterial+infection.">DCs in mouse models of intracellular bacterial infection.</a> Methods Mol Biol. 595, 319-329 (2010).</li><li>Conlan, J. W., North, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+are+essential+for+early+anti-Listeria+defense+in+the+liver,+but+not+in+the+spleen+or+peritoneal+cavity,+as+revealed+by+a+granulocyte-depleting+monoclonal+antibody.">Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody.</a> J Exp Med. 179, 259-268 (1994).</li><li>Czuprynski, C. J., Brown, J. F., Wagner, R. 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No conflicts of interest declared.

Listeria is one of the most widely used organisms to characterize host immune responses to intracellular bacteria6. The protocol presented here enables one to measure bacterial load and immune cell responses within the same tissue of a given mouse. This dual measurement of a particular tissue for each infected mouse provides for more robust comparisons within and between mouse cohorts (either representing different mouse strains or time points post-infection). While Listeria infection by oral administration could also be used to study immune responses in mice, infection by intravenous injection is often used because: 1) it ensures rapid and effective delivery to the bloodstream; 2) it results in a synchronized and consistent systemic infection; and 3) the Mus species harbors a mutation in the gene encoding the E-cadherin receptor, which limits Listeria infection by oral administration (this mutation affects Listeria's ability to bind the mouse E-cadherin receptor and to efficiently cross the epithelial lining of the gastrointestinal tract)16-18.
There are a few critical steps in this protocol. First, the Listeria inoculum stock should be generated from a fresh overnight HBA culture to ensure viability and virulence. Second, it is important to accurately determine the CFU concentration of the inoculum before and after injecting all mice to ensure that the CFU concentration does not differ greatly between the first and last mouse injected. Third, injection of Listeria into the tail vein must be consistent for all mice. Lastly, it is necessary to perfuse the liver to deplete non-resident leukocytes to ensure accurate measurement of immune cells within the liver and not leukocytes passing through in the peripheral blood. All of these steps, if not performed successfully, can result in unwanted variability for bacterial load and/or immune responses between individual mice infected with Listeria. 
Two limitations of this protocol are the investigator's skill for infecting mice by intravenous injection and the detection of bacterial load in tissues. If one person is injecting a large number of mice, Listeria viability (i.e. CFU concentration) may reduce over time once the frozen inoculum is thawed (e.g. &gt;2 hours between injecting first and last mouse). It is up to the investigator to determine how many mice she/he can inject before the inoculum is compromised. Another limitation of this method is that the Listeria load cannot be accurately measured below 100CFU/organ due to the relatively small amounts of cultured tissue homogenate (e.g. Figure 3 indicates that 100CFU/organ is the detection limit). To more accurately measure lower values for CFU/organ, a larger amount of the tissue homogenate can be cultured (up to 0.5mL per plate, multiple plates can be used) so a greater proportion of the tissue is sampled for detection of Listeria. If a Stomacher is not available, then alternative methods to homogenate the tissue, such as using a tissue homogenizer, can be used instead for steps 7.1-7.2. On a practical note, if space in the 37&deg;C incubator is limited for culturing tissue samples from infected mice, then HBA culture plates can be incubated at room temperature for 2-3 days (the colonies will grow slower at room temperature). However, room temperature should not be used when growing cultures for preparation of frozen Listeria stocks.
This protocol provides a basic approach for characterizing Listeria infection in mice and can be used with other Listeria strains besides EGD. In addition to the liver and spleen, single-cell suspensions from lymph nodes can also be generated. In either instance, these single-cell suspensions can be used for a variety of analyses, including measuring immune cell subsets, and in vitro stimulation of sorted immune cells. Once the basic techniques are mastered this protocol can also be modified to isolate Listeria-specific T cells19, characterize dendritic cells20, or perform immune cell depletion at certain time-points after infection21,22 to more thoroughly characterize the immune response in mice infected with Listeria.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Horse blood agar base No.2</td>
			<td>Oxoid</td>
			<td>CM0271</td>
			<td>Preparation of agar base for HBA plates</td>
		</tr>
		<tr>
			<td>Horse blood (defibrinated)</td>
			<td>Oxoid</td>
			<td>HB1000</td>
			<td>Add 5-10% to agar base</td>
		</tr>
		<tr>
			<td>Brain heart infusion (BHI) broth (10 mL)</td>
			<td>Oxoid</td>
			<td>TM456</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Brain heart infusion dehydrated media</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-well flat bottom plate</td>
			<td>BD Biosciences</td>
			<td>353072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainer</td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Small petri dish</td>
			<td>BD Biosciences</td>
			<td>351007</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stomacher 80 biomaster lab system</td>
			<td>Seward</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic Stomacher bags</td>
			<td>Sarstedt</td>
			<td>86 9924 530</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A3912</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.1% (w/v) bovine serum albumin in PBS</td>
		</tr>
		<tr>
			<td>Isotonic Percoll (33.75% Percoll in PBS)</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td>33.75mL Percoll, 3.75mL 10x PBS, 62.5mL 1x PBS makes 100mL isotonic Percoll</td>
		</tr>
		<tr>
			<td>TAC Buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>17mM Tris, 140mM ammonium chloride in distilled water</td>
		</tr>
		<tr>
			<td>FCS/EDTA buffer </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Fetal calf serum with 10mM EDTA</td>
		</tr>
		<tr>
			<td>FACS/EDTA buffer </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>FACS buffer + 5mM EDTA</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T6146</td>
			<td>0.4% (w/v) in PBS, filter sterilized</td>
		</tr>
		<tr>
			<td>2mL Cryovial</td>
			<td>Greiner Bio One</td>
			<td>121263</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27 gauge/1mL insulin syringe</td>
			<td>Terumo Medical Products</td>
			<td>SS10M2713</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo Medical Products</td>
			<td>NN-2516R (25G 5/8in) NN-2613R (26G 1/2in)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo Medical Products</td>
			<td>SS-01T (1mL), SS-053 (5mL), SS-10S (10mL)</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

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.

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 ...

measuring-bacterial-load-immune-responses-mice-infected-with-listeria

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Immunology and Infection

30.8K Views.  The University of Melbourne. 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.