Identification of the ferric iron utilization gene B739_1208 and its role in the virulence of R. anatipestifer CH-1.

Riemerella anatipestifer is an important bacterial pathogen in ducks and causes heavy economic losses in the duck industry. However, the pathogensis of this bacterium is poorly understood. In this study, a putative outer membrane hemin receptor gene B739_1208 in R. anatipestifer CH-1 was deleted to determine the relationship between iron uptake and virulence. The R. anatipestifer CH-1ΔB739_1208 mutants grew significantly more slowly than the wild-type bacteria in TSB liquid medium. Further characterization revealed that the R. anatipestifer CH-1ΔB739_1208 mutants were deficient in iron uptake. Animal experiments indicated that the median lethal dose of the wild-type RA-CH-1 in ducklings was 3.89×108, whereas the median lethal dose of the R. anatipestifer CH-1ΔB739_1208 mutant in ducklings was 5.68×109. The median lethal dose of the complementation strain in ducklings was 9.84×108. Additional analysis indicated that bacterial loads in the blood, liver, and brain tissues in the R. anatipestifer CH-1ΔB739_1208-infected ducklings were significantly decreased compared to those in the wild-type R. anatipestifer CH-1 infected ducklings. In a duck co-infection model with R. anatipestifer CH-1 and R. anatipestifer CH-1ΔB739_1208, the R. anatipestifer CH-1B739_1208 mutant was outcompeted by the wild-type R. anatipestifer CH-1 in the blood (P<0.002), livers (P<0.001) and brains (P<0.001) of infected ducks, indicating that B739_1208 gene expression provided a competitive advantage in these organs. Our results demonstrate that the B739_1208 gene is a virulence factor in R. anatipestifer CH-1.