Chicken embryo lethality assay for determining the lethal dose, tissue distribution and pathogenicity of clinical Enterococcus cecorum isolates from poultry

Enterococcus cecorum is a well-known component of the normal poultry intestinal microbiota and an important bacterial pathogen. Infections caused by E. cecorum have negative effects on the poultry production worldwide. In this study we used the SPF-chicken embryo lethality assay (ELA) to assess the pathogenic potential of E. cecorum. A total of 23 isolates were used: 19 clinical isolates from field outbreaks in different poultry groups (CB – broiler chickens, BB – broiler breeders, CL – layers, T– turkeys, W – waterfowl) and 4 commensal isolates. The cumulative mortality caused by all clinical isolates was higher (53.4%) than that of the commensals (38.9%). The highest mortality was induced by CB isolates (68.9%), followed by CL (60.4%), all chicken isolates (59.2%; CB, BB, CL), BB (45.8%), T (41.7%), non-chicken isolates (40.7%; T, W), and W isolates (39.8%). Most of the embryos that died, did die on the 1st day post-infection (dpi), except those infected with CB, CL (on 2 dpi). The median lethal dose (LD50) of E. cecorum ranged from 6.07 × 102 cfu/ml (CB isolates) and 1.42 × 104 cfu/ml (all clinical isolates) to 4.8 × 105 cfu/ml (commensal isolates). This study provides the first evidence of a wide tissue distribution and multiplication of E. cecorum in embryos. Dead embryos showed scattered petechiae, hemorrhages, aggregates of bacteria in blood vessels, multiple organ necrosis, and encephalomalacia. Our data indicate that surviving embryos were able to elicit innate immune response to infection. On the other hand, reisolation of viable bacteria from surviving embryos may suggest that E. cecorum could evade or resist immune mechanisms in order to persist in organs. Furthermore, body mass of surviving embryos was affected by the strain type, not the dose (bacterial concentration) used, and was lower for the infection with clinical strains. The results indicated the highest pathogenicity of clinical E. cecorum isolates from CB and CL flocks.