Abstract
Co-infection significantly influences disease severity. This study investigated the impact of co-infections with Escherichia coli (E. coli) O78 and Mycoplasma gallisepticum (MG), alone and in combination, on the pathogenicity of lowpathogenic avian influenza H9N2 in specific pathogen-free (SPF) chickens. Seventy one-day-old SPF chicks were divided into seven equal groups (G), where G1 is the control one, G2-G4 were infected with H9, MG, and E. coli, respectively, and G5-G7 were co-infected with MG-H9, E. coli-H9, and MG-E. coli-H9, respectively. The study monitored clinical symptoms, mortality rates, H9N2 hemagglutination inhibition antibody titers, viral shedding through qRT-PCR, and histopathological changes in experimentally infected groups. The findings revealed that the group co-infected with all three pathogens had the highest significant mortality rate (70%), with severe clinical symptoms, moderate histopathological changes in the trachea and lungs, along with the highest significant hemagglutination inhibition antibody titers (6.40±0.52, 7.30±0.67 log2) at 7 and 14 days post-infection, respectively. This group also demonstrated the highest viral shedding (3.53±0.01, 4.53±0.09, 3.60±0.05 log10 EID50/ml) at 2, 4, and 7 days post-infection, respectively, with significant differences, and the longest duration of H9N2 shedding (10 days post-infection). In summary, co-infection enhanced the pathogenicity of H9N2; furthermore, co-infection with E. coli O78 increased H9N2 pathogenicity more than co-infection with M. gallisepticum, and the combination of both bacteria resulted in the highest pathogenicity of the H9N2 virus.