Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes <4 weeks to complete, with the infection part taking only 3 d. We discuss the appropriate controls to use and potential adjustments needed for adapting the protocol for other microbial pathogens.
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J.J. was supported by Umeå University, the Swedish Research Council grant nos. K2011-56X-15144-08-6 and 621-2012-2451, the Knut and Alice Wallenberg Foundations, and European Research Council (ERC) starting grant no. 260764-RNAntibiotics.
After the chicken embryo has been isolated from the egg (see Figure 6a-c), the head of the chicken embryo is removed before embryo is placed on its back, in order to facilitate the liver extraction. Carefully open up the abdomen using a pair of forceps and extract the liver (see also Figure 6d). This step could prove difficult and might require some training on non-infected embryos. All experiments were performed in compliance with Swedish regulations.
By candling, the survival of a chicken embryo can be followed (see Figure 3b for setup). In this video, an alive chicken embryo is visualized (light torch on top). Please note the movement of the embryo and the well-defined capillaries (both thicker and thinner) inside the egg shell. Such capillaries are not observed in dead chicken embryos (compare Figure 3c and d). All experiments were performed in compliance with Swedish regulations.
Before perforation of the egg shell, the egg should be repeatedly disinfected using 70% ethanol. By a rapid movement, the egg shell is perforated using a pair of forceps (see also Figure 4a). The needle is inserted approximately 8 mm, through the chorioallantoic membrane at an angle of ∼45°C angle (see also Figure 4b). After the needle is removed, hot paraffin and tape is added onto the egg shell at the perforation site (see also Figure 4c and d). Remember to appropriately mark the egg for later identification. All experiments were performed in compliance with Swedish regulations.