Enterococcus cecorum are members of the normal microbiota in the intestinal tract of poultry, however, since the early 2000s virulent E. cecorum strains have emerged as a significant challenge in poultry production worldwide. Clinical outbreaks can have major economic impact through septicaemia, spondylitis, and bacterial osteomyelitis, which subsequently lead to locomotor disorders and increased mortality and condemnation rates have been reported. Strains are considered virulent when they are recovered from typical lesions in femur and free thoracic vertebra, whereas commensal or avirulent strains are generally isolated from the intestines and cloaca of healthy birds. The colonisation of the intestine followed by translocation and bacteremia is crucial to the pathogenesis of E. cecorum and development of lesions. The ability of E. cecorum to translocate from the intestinal tract to various tissues seems linked to virulence. This process of translocation and the interaction between bacterium and intestinal epithelium has not been studied in detail due to a lack of a suitable in vitro chicken model.
The aim of our study was to develop an in vitro/ex vivo model to investigate translocation and to differentiate between cloacal and lesion E. cecorum strains based on invasion and translocation. Models with increased cellular heterogeneity are more representative of the in vivo organ, however, typically, these more complex culture systems are more labour intensive and costly. The introduction of intestinal organoids has provided species- and organ-specific models which create more reliable intestinal responses than current in vitro options such as cell lines. A chicken organoid model with multiple villus-crypt structures was published in 2021, which uniquely maintains in vivo cellular diversity, polarity, and barrier function, incorporating cells of the lamina propria, while exhibiting an apical out orientation. These 3D chicken organoids are the most physiologically-relevant in vitro model available for studying intestinal health with the presence of an epithelial layer and an inner core containing the lamina propria, therefore, containing all cell lineages found in the intestinal tissue in vivo.
Materials & Methods
Experiments were performed using 18-day-old embryos of specific pathogen-free chicken bred at Royal GD. The generation of 3D intestinal organoids has been described previously, in brief intestinal villi are isolated by enzymatic digestion and after purification grown in a defined medium. The four E. cecorum strains used in this study are available at GD and were previously characterised by Manders et al.7 Two strains were isolated from cloacal swabs of healthy broiler reproduction animals (cloacal stains) and two strains were isolated from broilers with spondylitis and femur head necrosis (lesion strains).
After two days in culture the organoids were inoculated with 103–107 colony forming units (CFU) E. cecorum and after 3 or 6 hours post inoculation the number of bacteria were quantified by quantitative PCR.
Subsequently, an invasion assay was developed to determine translocation of bacteria. After inoculation with E. cecorum and incubation for 3 or 6 hours, the organoids were treated with a mixture of antibiotics, washed, and lysed with a detergent. The lysates were serially diluted and plated on Columbia agar supplemented with sheep blood and counted the next day.
