Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coli aroA mutant

In this study, an aroA-deletion avian pathogenic Escherichia coli (APEC) mutant (strain DE17 Delta aroA) and aroA and /uxS double deletion APEC mutant (strain DE17 Delta luxS Delta aroA) were constructed from the APEC DE17 strain. The results showed that as compared to DE17 Delta aroA, the virulence of DE17 Delta luxS Delta aroA was further attenuated by 200- and 31.7-fold, respectively, in ducklings based on the 50% lethal dose. The adherence and invasion abilities of DE17 Delta luxS Delta aroA and DE17 Delta aroA were reduced by 36.5%/42.5% and 25.8%/29.3%, respectively, as compared to the wild-type strain DE17 (p < 0.05 and 0.01, respectively). Furthermore, in vivo studies showed that the bacterial loads of DE17 Delta luxS Delta aroA were reduced by 8400- and 11,333-fold in the spleen and blood of infected birds, respectively, while those of DE17 Delta aroA were reduced by 743- and 1000-fold, respectively, as compared to the wild-type strain DE17. Histopathological analysis showed both that the mutants were associated with reduced pathological changes in the liver, spleen, and kidney of ducklings, and changes in DE17 Delta luxS Delta aroA-infected ducklings were reduced to a greater degree than those infected with DE17 Delta aroA. Real-time polymerase chain reaction analysis further demonstrated that the mRNA levels of virulence-related genes (i.e., tsh, ompA, vat, iucD, pfs, fyuA, and fimC) were significantly decreased in DE17 Delta aroA, especially in DE17 Delta luxS Delta aroA, as compared to DE17 (p < 0.05). In addition, the deletion of aroA or the double deletion of aroA and luxS reduced bacterial motility. To evaluate the potential use of DE17 Delta luxS Delta aroA as a vaccine candidate, 50 7-day-old ducklings were divided randomly into five groups of ten each for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17 Delta luxS, DE17 Delta aroA, and DE17 Delta luxS Delta aroA were 70.0%, 70.0%, 70.0, and 80.0% protected, respectively, after challenge with strain APEC DE17. The results of this study suggest that the double deletion of luxS and aroA attenuated APEC pathogenicity and DE17 Delta luxS Delta aroA was more appropriate for development of a future vaccine against avian colibacillosis than DE17 Delta aroA. (C) 2015 Elsevier Ltd. All rights reserved.