Some strains of Enterococcus cecorum can cause spondylitis and bacterial osteomyelitis. Translocation and bacteremia are pivotal to the pathogenesis and clinical disease. Virulence typing to distinguish extra-intestinal disease of lesion from cloacal strains remains difficult. We investigated if organoids can be applied to differentiate between E. cecorum strains that are more or less virulent. Floating chicken intestinal organoids combine the complex cell system of the gut with an easily accessible apical-out orientation. The organoids were treated with four E. cecorum strains that differ in original isolation, lesion, or cloacal, and bacterial load was determined after 3 and 6 h by quantitative PCR and bacterial plating. Independent of the inoculum dose or time post inoculation, DNA levels of E. cecorum marginally differed between the strains. To determine if this was caused by adherence of bacteria to the epithelial cells, an invasion assay was developed. The organoids were inoculated with the different E. cecorum strains and after 3 or 6 h treated with an antimicrobial mixture, lysed, and quantified by bacterial plate counting. Significantly higher (p < 0.0001) numbers of bacteria isolated from lesions invaded the organoids compared to cloacal strains in a dose-dependent manner. Higher numbers of bacteria isolated from lesions invaded the organoids compared to cloacal strains in a dose-dependent manner. This study is a major step in the development of a model to study the interaction between E. cecorum and the chicken host and a model to test novel intervention strategies to prevent translocation of bacteria.

Enterococcus cecorum infections can be experimentally reproduced after oral inoculation. Co-infections of E. cecorum with other avian pathogens might increase the proportion of broilers with E. cecorum infections. The aim of the study was to examine via which infection route E. cecorum is capable of causing E. cecorum infections and which co-infections exacerbate E. cecorum infections. Two experiments, each with 12 groups, were conducted. Per group 40 SPF broilers were inoculated intravenously, orally or via aerosol with E. cecorum alone, a negative control group was included and in eight groups the effect of co-infections was studied. In experiment 1, infectious bronchitis virus (IBV), Newcastle disease virus (NDV) vaccine, reovirus, chicken anaemia virus (CAV) were inoculated prior to oral or aerosol challenge with E. cecorum. While in experiment 2, E. cecorum was given after administration of dexamethasone or inoculation of CAV or combinations of IBV or NDV with Mycoplasma synoviae (M.s.). Typical E. cecorum lesions were reproduced via all applicated infection routes. The proportion of lesions were highest in the intravenous inoculated groups, respectively 0.25 (95% Confidence Interval (CI):0.127-0.412) and 0.275 (95% CI:0.146-0.439) in experiment 1 and 2. More lesions were observed in experiment 2, while the proportion of lesions after oral and aerosol inoculation did not differ from each other in both experiments. The proportion of lesions was not exacerbated after co-infections of E. cecorum with various avian pathogens. Only the proportion of E. cecorum reisolations was exacerbated after inoculation with CAV or M.s. in combination with IBV or NDV.

Reporte de caso- Brote de endocarditis asociada a Enterococcus hirae en reproductoras jóvenes de la línea de hembras. Las infecciones por enterococos pueden provocar una enfermedad aguda y crónica. Las lesiones, como osteomielitis, artritis o endocarditis, dependen de la especie de Enterococcus involucrada. En este informe de caso, se presenta un brote de endocarditis asociada a Enterococcus hirae que afectó a reproductoras pesadas jóvenes. En una granja de crianza con 46,500 reproductoras pesadas, se observó un ligero aumento de la mortalidad (1.02%–1.42%) dentro de la línea de hembras entre las semanas 2 y 6 posteriores a la eclosión. Entre los días 12 y 47 después de la eclosión, se examinaron mediante necropsia 256 aves y se tomaron muestras bacteriológicas. En 191 (51.8%) de los pollos de la línea hembra examinados, se observó endocarditis en al menos una de las válvulas auriculoventriculares, mientras que no se diagnosticó endocarditis en pollos de la línea macho (n = 65). Se aisló Enterococcus hirae del 64.4% de las muestras de médula ósea y de dos muestras de corazón tomadas de pollos de la línea hembra. Enterococcus hirae no se aisló de muestras tomadas de la línea macho. Además, los títulos de anticuerpos naturales (inmunoglobulina G) fueron significativamente más altos en el suero de la línea hembra en comparación con la línea macho. Este caso ofrece una visión general de las lesiones macroscópicas y los hallazgos bacteriológicos de E. hirae que afecta solo a la línea hembra de reproductoras de pollos de engorde.

In ovo vaccinations have been widely used in the poultry industry over the past three decades. During vaccination, various factors, including bacterial contamination of embryonated eggs, can negatively affect hatchability, chick quality and first-week performance. The aim of this study was to assess the effect of inoculation with different bacteria at incubation day 18 on hatchability, chick quality and first-week mortality. Furthermore, the effects of different bacterial doses on the same parameters were assessed. First, inoculation strains were selected by isolating bacteria from unhatched, in ovo-vaccinated eggs in commercial hatcheries. A total of 23 bacteria species were isolated, with E. coli and E. faecalis being the most commonly found species in eggs from all hatcheries. The virulence of ten E. coli and ten E. faecalis isolates was assessed using an embryo lethality assay. Subsequently, the most virulent strains of E. coli and E. faecalis were selected for an animal experiment. Eighteen-day incubated embryonated broiler eggs were inoculated with 0.1 ml of bacteria suspension or peptone physiological saline, using 36 eggs per group. Three different bacterial doses (10², 10⁴ or 10⁶ colony-forming unit/egg) were used. Hatching rate, time of hatch, chick length, and Pasgar score were determined at the end of the incubation process and mortality, time of death, and chick weight in the first week of life. Hatchability did not significantly differ between the different doses when E. faecalis was inoculated, ranging from 56 to 69 %. First-week mortality in these groups ranged from 36 to 86 %, while chick quality did not differ from the control groups. E. coli was pathogenic at all tested doses, with no chicks hatching in any of these groups. In all groups (E. coli and E. faecalis), severe drops in hatchability and increased first-week mortality were observed. Therefore, strict hygiene measures should be implemented in hatcheries during in ovo vaccination to prevent contamination of eggs with low numbers (<10² cfu/egg) of virulent bacteria, such as E. coli and E. faecalis.

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.

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a disease of major economic importance to the broiler industry. This study aimed to investigate genetic variation in susceptibility to colibacillosis by comparing four pure broiler breeder lines and their commercial four-way cross offspring. Three consecutive experiments were performed assessing mortality, growth retardation and mean lesion scores (MLS) after E. coli challenges. In the first experiment, birds were challenged intratracheally with a high dose of E. coli at 8 days of age. All pure lines showed significantly higher mortality rates (54.2-80.6 %) compared to the commercial line (20.9 %). The second experiment tested the most and least susceptible pure lines from Experiment 1, alongside the commercial line. Low, medium, and high E. coli doses were inoculated at the same inoculation day as Experiment 1. A clear dose-dependent effect on mortality and growth was observed across all lines, confirming that disease severity is linked to challenge dose. The third experiment compared the most susceptible pure line and the commercial line using a dual-infection model. Prior to the E. coli-inoculation, an infectious bronchitis virus (IBV) vaccine strain was administered. The results were in line with the first two experiments, the commercial line performed better on all parameters compared to the tested pure line. No significant differences in susceptibility were found between males and females in any experiment. The commercial broilers, a four-way cross of the tested pure lines, demonstrated superior resistance to colibacillosis compared to the pure lines in all experiments. Among the pure lines, genetic differences in susceptibility to colibacillosis were observed. These differences indicate that genetic selection for resistance to colibacillosis might be feasible.