Novel insights into avian pathogenic Escherichia coli pathogenesis and host response using bioluminescent imaging

Background: Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis, a common bacterial disease in the poultry industry with economic importance, unresolved pathogenesis, and potential zoonotic risk. Despite extensive research on disease pathogenicity, little is known about disease progression and host-pathogen interaction. Objectives: a) assess if bioluminescence imaging could track the progression of APEC in chicken embryos in real-time and non-invasively; b) evaluate the enhanced light intensity generated by ilux2-APEC after being injected into a chicken embryo; c) investigate the pathological and microbial sequential progression of colisepticaemia in layers, and transmission of E. coli into eggs using ilux2-APEC. Methods: For in ovo inoculation, genetically modified E. coli strains luxABCDE-E. coli PA14/17480/5-ovary and ilux2-E. coli PA14/17480/5-ovary, along with the parent strain, were used. Afterwards, eggs were imaged daily for bioluminescent detection and quantification up to 5 days post-infection (dpi). For the in vivo infection model, 48 hens were allocated into three groups, and were intratracheally inoculated with ilux2-E. coli PA14/17480/5-ovary, E. coli PA14/17480/5-ovary, or PBS. Results: In the in ovo infection model, embryo lethality was 80-100%, and different bioluminescence patterns were observed when tracking lux-tagged APEC. The main pattern showed increasing bioluminescence over time. In ovo (intact egg) and ex ovo (after removing eggshell) bioluminescence strongly correlated. Higher bioluminescence values were obtained from ilux2-E. coli PA14/17480/5-ovary infected embryos compared to luxABCDE-E. coli PA14/17480/5-ovary . In the in vivo infection model, both strains caused typical clinical signs and lesions of colibacillosis similar to field outbreaks. Based on lung histopathology, colisepticaemia progression was divided into four disease stages characterized histologically by heterophils (1-3 dpi), mixed cells (6 dpi), pyogranuloma (9 dpi), and convalescence (16 dpi), respectively. As disease progressed, bacterial colonization in host organs decreased, as indicated by measures of bacterial bioluminescence, bacteriology, and quantitative immunohistochemistry. Additionally, immunofluorescence, immunohistochemistry, and bacteria re-isolation confirmed this pattern for E. coli colonize in hens' reproductive tracts and reach egg contents. Conclusion: The bioluminescence imaging system with ilux2 operon provides new insights into colisepticaemia pathogenesis by characterizing microbial growth dynamics, host-pathogen interaction during infection, and bacteria transmission to table eggs, which have significant implications for poultry health and food safety.

• VetImaging