Measuring Bacterial Load and Immune Responses in Mice Infected with <em>Listeria monocytogenes</em>

Abstract

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen 1 . Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection 2 . After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8α + dendritic cells and Kupffer cells 3,4 . . 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 infection 6,[8][9][10][11][12][13][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 strain 15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to β-hemolysis 1 (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. Video LinkThe video component of this article can be found at https://www.jove.com/video/3076/ Protocol 1. Culturing and long-term storage of Listeria monocytogenes (Listeria)1. Make or purchase pre-made horse blood agar (HBA) plates. Dry plates prior to use by pre-incubating at 37°C overnight or placing uncovered in a laminar flow cabinet for 1 hour. 2. Obtain viable Listeria from one of the following: an infected mouse tissue homogenate, a lyophilized stock, a frozen glycerol stock, or a colony from a recent Listeria culture (i.e. less than one-week old and not sub-cultured more than 10 generations). All Listeria should be obtained using a sterile inoculating loop and employing aseptic techniques for all methods described in this protocol. 3. Streak Listeria on a HBA plate as shown in Figure 1 using a sterile ino...

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