Embryonated Eggs as an Alternative Infection Model To Investigate Aspergillus fumigatus Virulence

Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the “gold standard” for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity.Aspergillus fumigatus is a ubiquitous mold with the ability to cause life-threatening disease in immunocompromised human patients (19). Despite the development of novel antimycotics, the lethality of invasive aspergillosis in human patients is still high and can exceed 50% (14, 57). Thus, a better understanding of the infectious process and the fungal factors involved in pathogenesis is essential for development of more effective treatments. One approach to identify virulence factors is to study fungal gene deletion mutants in appropriate infection models. The most commonly used infection models for A. fumigatus are laboratory rodents, especially mice. These models are well characterized and have been critical for understanding host-pathogen interactions, as well as for developing better therapeutic approaches (11). However, ethical considerations and legal restrictions limit the use of mammals for infection studies. Additionally, animal experiments are costly and require specialized facilities and specially trained personal, which further limits the availability of mammalian models to researchers. Therefore, several alternative infection models for fungal pathogens have been developed in recent years, ranging from cell lines and tissue cultures to invertebrate hosts. Insect models, like the fruit fly (Drosophila melanogaster) (28, 29) and larvae of the greater wax moth (Galleria mellonella) (21, 30, 37, 42, 43), have been successfully used to investigate virulence traits of A. fumigatus and the efficacy of antimycotic treatment. However, these models are limited compared to mammalian hosts; the limitations include, for example, the low temperature required for experiments, the route of infection, and certain aspects of the immune response. One possible way to bridge the gap between insect and mammalian hosts is to use embryonated eggs as an alternative infection model.Birds of all ages are susceptible to aspergillosis, and this disease has been described for a variety of avian species, both birds in captivity and wild birds (54). Aspergillosis outbreaks can cause significant economic losses in the poultry industry (41) and have prompted the development of vaccination strategies to prevent Aspergillus infections (46). The avian immune response to Aspergillus infections is similar to the mammalian response with respect to the beneficial effect of a type 1 response, the role of macrophages and heterophils (avian immune cells similar to mammalian neutrophils), and the production of specific antibodies (10, 27, 33). Although these features suggest that birds could be an interesting and potential animal model for studying aspergillosis (11, 47), the same ethical, legal, financial, and logistical restrictions that apply to experiments with mammals apply to experiments with birds. However, embryonated bird eggs could provide an alternative model, which would fulfill the demand for refinement of animal models. Fertilized eggs are readily available from commercial breeders at low cost, are easy to handle, and require little specialized equipment and no specialized facilities or personal. Since embryonated eggs have been successfully used to study virulence features of various bacteria (2, 55) and the fungal pathogen Candida albicans (16, 17), we investigated the potential of embryonated eggs as an alternative model for studying the virulence of A. fumigatus.