Novel insights into the virulence and pathogenesis of the emerging bacterium Enterococcus cecorum

Enterococcus cecorum is a Gram-positive bacterium that is a normal inhabitant of the gastrointestinal tract of chickens. Over recent decades, however, there has been an increasing incidence of E. cecorum-associated infections, particularly in broiler chickens. These infections are characterized by severe lesions, including spondylitis, pericarditis, and femoral head necrosis. Affected birds may develop lameness or leg paralysis, resulting in impaired welfare and substantial economic losses in the poultry industry. This dissertation aimed to elucidate the virulence of E. cecorum and to investigate the mechanisms of pathogenesis contributing to disease development. Specifically, the natural routes of infection and the effects of co-infections with other avian pathogens were studied. Furthermore, lesion-derived isolates were compared with commensal cloacal isolates to identify traits associated with virulence. Animal experiments using specific-pathogen-free broilers demonstrated that both oral and aerosol exposure can induce typical E. cecorum lesions. Co-infections with avian pathogens, including infectious bronchitis virus (IBV), Newcastle disease virus (NDV), reovirus, Mycoplasma synoviae, and chicken anaemia virus (CAV), did not significantly increase lesion incidence. Only combinations of E. cecorum with CAV or M. synoviae in conjunction with IBV or NDV resulted in higher reisolation rates of E. cecorum. Embryo lethality assays are widely employed to assess bacterial virulence. However, the most suitable inoculation route for distinguishing between virulent and non-virulent E. cecorum strains in this assay had not been established. Therefore, an initial study was performed to evaluate the success rates of inoculations targeting different compartments of the embryonated egg. Inoculation success varied depending on both the embryonic stage and the route of administration. Subsequent experiments demonstrated that albumen inoculation was optimal for discriminating lesion from cloacal isolates based on embryo mortality. Given that albumen possesses antimicrobial properties, with lysozyme as a principal component, the resistance of E. cecorum isolates to albumen and lysozyme was assessed. Lesion isolates exhibited reduced sensitivity to albumen and increased resistance to lysozyme compared to cloacal isolates, indicating an enhanced capacity for survival within the host. Assessment of lysozyme resistance may therefore represent a useful screening method for virulence. Translocation and bacteraemia are critical events in the pathogenesis of E. cecorum infections. Using chicken intestinal organoids, lesion-derived isolates were shown to invade the organoids more efficiently than cloacal isolates in a dose-dependent manner. This organoid model provides a relevant platform for investigating host–pathogen interactions and evaluating potential intervention strategies. In conclusion, this dissertation demonstrates that both oral and aerogenous exposure to E. cecorum can induce typical lesions, and that lesion isolates display enhanced virulence traits across multiple experimental models. The approaches developed herein, including embryo lethality testing, lysozyme resistance assays, and intestinal organoid invasion model, provide valuable tools for advancing research on E. cecorum virulence and pathogenesis. Collectively, these findings enhance the understanding of this emerging poultry pathogen and support the development of preventive and diagnostic strategies.