| Avian colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Escherichia coli (APEC), including embryo death, omphalitis septicaemia, granuloma, peritonitis/yolk, panophthalmitis, air sacculitis, avian cellulitis, swollen head syndrome, salpingitis, synovitis, pneumonia and yolk infection. So far, this disease has been an important bacteriosis that has became a heavy threat to and caused significant economic losses in poultry industry. APEC has a specific iron assimilation system that consists of siderophore, aerobactin and outer membrane receptor proteinencoded by iucA、 iucB、 iucC、iucD and iutA. In order to master the pathogenic mechanism and prevention or control measures of the disease, and to understand the biological characteristics of the mutants, E058iucCiucD, E058iucD, E058iucDiutA mutants were developed.In this study, genes in APEC serotype02strain E058chosen for deletion were iucC, iucD and iutA based on the technology of Red Homologous Recombination. A mutant was constructed and named as E058iucD, and two double mutants (E058iucCiucD and E058iucDiutA) were also constructed. The plasmid of pGEX-6P-1-iucD was electroporated into E058iucD to get the revertant of E058iucD.The results of the Southern blot showed zeocin resistance gene or Kanamycin gene was singly inserted in pAPEC-02-like plasmid of E058iucCiucD, E058iucD, E058iucDiutA. Three mutants were disrupted in the expression of the target gene or genes, while the upstream gene and the downstream gene of the target were expressed normally. For example, E058iucCiucD did not transcript the gene of iucC and iucD, but iucB (the upstream gene of iucC) and iutA (the downstream gene of iucD) were transcripted normally. E058iucCiucD, E058iucD and E058iucDiutA were not able to produce aerobactin, whereas E058and ReE058iucD were able to do. The growth curves of E058iucCiucD, E058iucD, E058iucDiutA were similar to that of their parental strain in LB broth. In the out-competition test in vivo, the competition index (CI) values of E058iucCiucD and E058for24h post-challenge in heart, liver, spleen, lung and kidney were0.80,0.30,0.36,0.39,0.50. The CI values of E058iucD and E058for24h post-challenge were0.02,0.31,031,0.35,0.46respectively. The CI values of E058iucDiutA and E058for24h post-challenge in heart, liver, spleen, lung and kidney were0.30,0.05,0.06,0.41,0.08respectively. The CI values of mutanted strains and the wild-type strain in these organs varied from0.1～1, which indicated E058iucCiucD、 E058iucD and E058iucDiutA were all lightly attenuated compared with their parental strain. In the challenge model with a sigle strain, numbers of E058iucCiucD recovered from heart, liver, spleen, lung and kidney of the challenged birds at24h post-challenge were4.24±0.37,4.75±0.46,4.97±0.22,4.85±0.35,4.46±0.69(lg) respectively. At the same time, for E058iucD, those were2.92±0.29,4.16±0.37,4.49±0.75,4.08±0.44,3.98±0.98respectively, and for E058iucDiutA, those were3.89±1.12,4.68±0.91,4.54±0.64,4.77±0.97,3.74±2.17, respectively. Meanwhile, those for wild-type strain E058were4.62±0.95,4.77±0.70,5.20±0.95,5.38±0.88,4.62±1.06respectively. It seemed that the bacterial loads of all three mutants in the tissues of challenged birds were obviously decreased compared with their parental strain. These results indicated that the iucC, iucD, iutA genes related virulence factors were important for the pathogenesis of APEC E058. |
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