Objective: To find the primary function of aes-31 fragment through construction of defined mutation of Avian Pathogenic Escherichia coli strain E058 and animal experiments.
Methods: The fragment of aes-31 was generated by PCR and cloned into pGEM-T-easy vector. A resultant suicide vector containing the aes-31 fragment named pMEG375-aes-31 was constructed and transformed to a receptor strain SM10. Then recombinant strain SM10 was hybridized with E058 strain in solid state. Mutant derivatives of strain E058 were generated by homologous recombination and were named E058 (delta aes-31). The 50% lethal dose (LD50) of E058 and E058 (delta aes-31) in commercial day-old chickens experimentally inoculated via intratrachea were 10(4.3) CFU and 10(3.5) CFU, respectively. The same way was used to inoculate with 10(8) CFU to obtain the pathogenic ability of E058 and E058 (delta aes-31) in 35-days-old SPF chickens. In the chicken challenge model,the mutant was tested to determine the individual function for virulence and persistence in 2-week-old SPF chicks.
Results: The pathogenicity test for E058 strain and E058 (delta aes-31) strain showed that the mutant had a higher mortality (75%) to 35-day-old specific pathogen-free (SPF) chicks than that of E058 (62.5%). In the chicken challenge model,there was no obviously CFUs difference in blood and lung in chicks of E058 group and E058 (delta aes-31) group 6 hours after inoculation. After 24 hours there was obvious CFUs difference in heart, liver, spleen, lung and blood in chicks of E058 group and E058 (delta aes-31) group. After 48 hours, there was also obvious CFUs difference in heart, liver and spleen in chicks of E058 group and E058 (delta aes-31) group E058 (delta aes-31) had a trend of increasing virulence in chicks.
Conclusion: Aes-31 might be associated with negative regulatory gene for E058 virulence and its actual function needed further study.