Name: measuring bacterial load and immune responses in mice infected with listeria monocytogenes
Uploaded: 2011-08-09T13:00:00
Duration: 12 min 50 s
Description: Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

लेखकों के लिए सलाह और अभिकर्मकों के लिए अन्ना Walduck, क्रिस्टीना चीयर्स, स्टुअर्ट Berzins, डेल गॉडफ्रे, Yifan Zhan और जोनाथन Wilksch धन्यवाद देना चाहूंगा. यह काम किशोर मधुमेह अनुसंधान फाउंडेशन (1-2008-602) और ऑस्ट्रेलियाई राष्ट्रीय स्वास्थ्य और चिकित्सा अनुसंधान परिषद (575,552) द्वारा वित्त पोषित किया गया था. एनडब्ल्यू एक ऑस्ट्रेलियाई स्नातकोत्तर पुरस्कार द्वारा समर्थित है. ओ ऑस्ट्रेलियाई राष्ट्रीय स्वास्थ्य चिकित्सा अनुसंधान परिषद से एक आरडी राइट फैलोशिप द्वारा समर्थित है.

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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=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. D., Steinberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+antigranulocyte+monoclonal+antibody+RB6-8C5+prevents+expression+of+acquired+resistance+to+Listeria+monocytogenes+infection+in+previously+immunized+mice.">Administration of antigranulocyte monoclonal antibody RB6-8C5 prevents expression of acquired resistance to Listeria monocytogenes infection in previously immunized mice.</a> Infect Immun. 62, 5161-5163 (1994).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

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> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियाँ </th></tr><tr><td> हार्स रक्त अगर आधार No.2 </td><td> Oxoid </td><td> CM0271 </td><td> एचवीए प्लेटों के लिए अगर आधार की तैयारी </td></tr><tr><td> हार्स रक्त (Defibrinated) </td><td> Oxoid </td><td> HB1000 </td><td> अगर आधार करने के लिए 5-10% जोड़ें </td></tr><tr><td> ब्रेन दिल (भी) जलसेक शोरबा (10 एमएल) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> ब्रेन दिल जलसेक निर्जलित मीडिया </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96 अच्छी तरह से फ्लैट नीचे की थाली </td><td> बी.डी. बायोसाइंसेज </td><td> 353072 </td><td></td></tr><tr><td> 70 सुक्ष्ममापी सेल झरनी </td><td> बी.डी. बायोसाइंसेज </td><td> 352350 </td><td></td></tr><tr><td> लघु पेट्री डिश </td><td> बी.डी. बायोसाइंसेज </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 biomaster प्रयोगशाला प्रणाली </td><td> सेवार्ड </td><td></td><td></td></tr><tr><td> प्लास्टिक Stomacher बैग </td><td> Sarstedt </td><td> 86 530 9924 </td><td></td></tr><tr><td> गोजातीय सीरम albumin </td><td> सिग्मा </td><td> A3912 </td><td></td></tr><tr><td> FACS बफर </td><td></td><td></td><td> 0.1% (w / v) पीबीएस में गोजातीय सीरम albumin </td></tr><tr><td> Isotonic Percoll (पीबीएस में 33.75% Percoll) </td><td> जीई हेल्थकेयर </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10x पीबीएस, 62.5mL 1x पीबीएस 100ml isotonic Percoll बनाता है </td></tr><tr><td> टीएसी बफर </td><td></td><td></td><td> 17mm Tris, आसुत जल में 140mm अमोनियम क्लोराइड </td></tr><tr><td> FCS / EDTA बफर </td><td></td><td></td><td> 10mm EDTA के साथ भ्रूण बछड़ा सीरम </td></tr><tr><td> FACS / EDTA बफर </td><td></td><td></td><td> FACS + बफर 5mm EDTA </td></tr><tr><td> Trypan नीले </td><td> सिग्मा </td><td> T6146 </td><td> पीबीएस (w / v) में 0.4%, फिल्टर निष्फल </td></tr><tr><td> 2ml Cryovial </td><td> Greiner एक बायो </td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL इंसुलिन सिरिंज </td><td> Terumo मेडिकल उत्पाद </td><td> SS10M2713 </td><td></td></tr><tr><td> सुई </td><td> Terumo मेडिकल उत्पाद </td><td> NN 2516R (25g 5/8in) NN 2613R (26G 1/2in) </td><td></td></tr><tr><td> सिरिंज </td><td> Terumo मेडिकल उत्पाद </td><td> एसएस-01T (1ml), एस एस 053 (5ml), एसएस 10S (10ml) </td><td></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

लिस्टेरिया monocytogenes प्रतिरक्षा प्रतिक्रिया और आनुवंशिक संवेदनशीलता का अध्ययन चूहों में बैक्टीरिया intracellular के लिए एक मॉडल जीव है. इस विधि में एक जीवाणु लोड को मापने के लिए और FACS विश्लेषण के लिए चूहों से जिगर और तिल्ली के एकल कक्ष निलंबन उत्पन्न लिस्टेरिया संक्रमण के कारण प्रतिरक्षा कोशिकाओं में परिवर्तन का निर्धारण करने में सक्षम बनाता है.

से संक्रमित चूहों में बैक्टीरियल लोड और इम्यून प्रतिक्रियाएँ माप लिस्टेरिया monocytogenes</em

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

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Immunology and Infection

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

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Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Responses

साथ प्रायोगिक संक्रमण<em&gt; लिस्टेरिया monocytogenes</em&gt; का अध्ययन होस्ट इंटरफेरॉन-γ जवाब के लिए एक मॉडल के रूप

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

अलगाव साल्मोनेला typhimurium-मैक्रोफेज से युक्त Phagosomes का

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

संक्रमित Ifnar1 के Immunoprivileged अंगों में Zika वायरस-विशिष्ट टी-सेल प्रतिक्रियाओं का मूल्यांकन-चूहों/

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes Infection of the Brain

लिस्टिरिया monocytogenes मस्तिष्क का संक्रमण

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