Embryonated Chicken Eggs as Alternative Infection Model for Pathogenic Fungi
Embryonated eggs have been used as infection models for decades in virology and bacteriology. However, they can also be used as an attractive alternative infection model for studying fungal pathogenesis. Here, we discuss some general aspects which need to
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1. Introduction Infection models are essential tools in investigating host-pathogen interaction, pathogenicity mechanisms, virulence attributes of pathogenic fungi, and therapy studies. Thus, in vivo models, mainly using laboratory rodents, such as mice, have been developed for many microbial pathogens. These models have been critical for understanding host-pathogen interactions as well as for developing better therapeutical approaches (1) and represent the current “gold standard” of in vivo virulence testing. However, ethical considerations and legal restrictions limit the use of mammals for infection studies. Further limitations arise from the requirement for specialized animal facilities and personal, as well as cost. Alternative in vivo infection models using lower animals have therefore been developed in recent years. Embryonated chicken eggs provide an alternative in vivo infection model which requires little specialized equipment, is easy to
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_34, © Springer Science+Business Media, LLC 2012
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handle and is available at lower costs than murine models. This model has been successfully used in studying virulence determinants of viruses and bacteria (2, 3), as well as Candida albicans (4, 5), during recent decades. We have recently investigated the suitability of embryonated chicken eggs, infected on the chorioallantoic membrane (CAM), to determine the virulence potential of defined gene knockout mutants of Aspergillus fumigatus (6). We found the model to be highly reproducible with good correlation to results obtained from infection studies in mice. In addition to measuring virulence in survival experiments, the chicken embryo model allows quantification of fungal burden, histological assessment of invasiveness, and quantification of the host immune response on the transcriptional level (6). While murine models undoubtedly remain the “gold standard” for assessing strain virulence, the embryonated egg model can be useful as a screening tool to define the most interesting mutants for subsequent testing in mice. In this chapter, we describe the handling and preparation of embryonated eggs for infection, infection of the CAM, determination of embryonic viability, and collection of samples for downstream analyses. We have successfully used this method not only for A. fumigatus (6) but also for other pathogenic fungi, e.g. C. albicans (unpublished data) and Lichtheimia spp. (unpublished data) (see Note 1).
2. Materials 2.1. Preparation of Eggs for Infection
1. Fertilized chicken eggs (see Note 2). 2. Egg incubator set to 37.6°C and 60% relative humidity (see Note 3). Specialized egg incubators (e.g. from Grumbach, Germany) additionally allow automated turning of eggs. Ideally, the incubator should be placed in a room which can be darkened to allow easier evaluation of viability by candling. 3. A suitable tray for eggs.
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