| Avian pathogenic Escherichia coli(APEC)is an important extraintestinal pathogenic Escherichia coli(ExPEC),which causes a variety of local or systemic acute and chronic infectious diseases in avian and leads to huge economic losses to the poultry industry.As APEC shares some virulence factors with ExPEC,it is important to study the pathogenicity of APEC.At present,the main epidemic serotypes of APEC are O1,O2 and O78 in the world.By analyzing the whole genome sequence of APEC strain DE205B(O2:K1),a complete prophage,named phiv205-1,was predicted in the genome of DE205B.In order to investigate the effect of this prophage on the virulence and environmental tolerance of DE205B,the prophage mutant strain was constructed by using the Red homologous recombination system,and the biological characteristics of the wild-type(WT)strain DE205B and the mutant strain DE205B?phiv205-1 were studied.1.The effect of prophage phiv205-1 on pathogenicity of DE205BThe results of cell adhesion and invasion test showed that compared with the WT strain,the adhesion and invasion abilities of the mutant strain to the chicken embryo fibroblast(DF-1)decreased by 41.88%(P<0.01)and 40.94%(P<0.05)respectively.The biofilm formation ability of the mutant strain decreased by 52.38%(P<0.001)compared with the WTstrain.The colonization experiment in vivoshowed that the bacterial load of DE205B ? phiv205-1 in lungs and brains decreased by 1.6 times(P<0.05)and 4.8 times(P<0.001)respectively compared with the WT.The virulence of the WT strain and the prophage deletion mutant were measured by tracheal inoculating theone-week old healthy chicks.The results showed that the LD50 of the WT and mutant strain on chicks were 3.86 × 104 CFU and 3.13 × 105 CFU per chick respectively,indicating that the virulence of DE205B decreased after the deletion of phiv205-1.In conclusion,the prophage phiv205-1 playedan important role in the process of cell adhesion and invasion,biofilm formation and colonizationin vivo of host bacteria DE205B,and finally promoted the pathogenicity of DE205B.2.The effect of prophage phiv205-1 on the environmental tolerance of DE205BIn order to test the effect of prophage phiv205-1 on the environmental tolerance of DE205B,the environmental tolerance tests were carried out on the WT strain and the mutant strain respectively.The results showed that compared with the WT strain,the survival rate of the mutant strain DE205B?phiv205-1 in an acidic environment(pH4.0;30 min),hypertonic environment(2.4mol/L NaCl;60.min)and oxidative stress environment(10mmol/L H2O2;15 min)was decreased by 56.62%(P<0.001)?11.41%(P>0.05)and 91.78%(P<0.001),indicating that the prophage phiv205-1 was contributed to DE205B to resist the adverse environment In order to study the mechanism of prophage enhancing the antioxidant stress of APEC,some genes were selected,the expression level of which in the oxidative stress environment were then detected.The results showed that the transcription levels of genes 2971,2989 and Erf significantly increased under oxidative stress.Single gene deletions of ?2971??2989 and ?Erf were further constructed.The results showed that compared with WT strain,the survival rates of mutant strains ?2971??2989 and ?Erf were reduced by 89.32%(P<0.001)?67.79%(P<0.05)and 71.29%(P<0.01)under oxidative stress,indicating that the genes 2971,2989 and Erf encoded by prophage played an important role in the tolerance of DE205B to oxidative stress. |
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