Construction and Characterization of Avian Pathogenic Escherichia coli Mutants with iro and/or tsh Gene Mutation

Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive hybridization (SSH) and selective capture of transcribed sequences (SCOTS) in avian pathogenic Escherichia coli (APEC) isolates. To get more insights in the distribution and the occurrence of the iroC and tsh genes, 243 avian E. coli isolates were examined for the presences of these genes. Amongst 243 avian E. coli isolates, iroC gene was present in 84.4% strains (205/243). Of the 205 iroC-positive isolates, iroC gene was found in 184 (89.8%), 18 (8.8%) and 3 (1.5%) isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 (87.4%), 21 (12.6%) and 0 (0%) isolates with high, intermediate and low pathogenicity, respectively. Amongst tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene (P<0.01). Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragment of iroB and 603-bp fragment of the iroN were amplified by PCR, respectively. Both these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector combined with the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm, and a resultant suicide vector combined with the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037 (Δiro), E037 (Δtsh) and E037 (ΔiroΔtsh). The 50% lethal dose (LD50) of E037, E037 (Δiro), E037 (Δtsh) and E037 (ΔiroΔtsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined as 105.6, 108.4, 109.0 and 109.5CFU, respectively. In the chicken challenge model, the mutants were evaluated on the individual role of these genes for virulence and persistence in chickens. The result indicated that Iro system and Tsh were important in the pathogenicity of APEC.