| Avian pathogenic Escherichia coli(APEC)is a common pathogen causing poultry disease.It can cause avian colibacillosis characterized by sepsis and extensive tissue inflammation,which seriously harms the poultry industry and causes huge economic losses.The abuse of antibiotics has led to a large increase in APEC's multi-drug resistant strains,and the prevention and treatment methods relying on antibiotics are facing great challenges.The use of non-pharmaceutical methods to prevent and control APEC is an inevitable trend in future development,and prevalence of poultry colibacillosis,reduce pollution of poultry products,and ensure food safety.Outer Membrane Vesicless(OMVs)are a nano-scale(20-200 nm)spherical phospholipid double-layered vesicle structure secreted by gram-negative bacteria,which encapsulate various components of the bacteria,such as the outer membrane proteins,periplasmic proteins,phospholipid molecules,lipopolysaccharides,nucleic acids,etc.OMVs,having good immunogenicity and adjuvant activity,can effectively stimulate the host immune system and therefore possess the potential to be developed into vaccine.In the early development of vaccines based on OMVs,it was found that the production of OMVs secreted by APEC wild-type strains during growth was low.Studies have reported that the tolA gene located on the Tol-Pal system plays a role in maintaining the stability of the bacterial outer membrane and affecting the production of OMVs.For example,the deletion of tolA can significantly enhance the production of Shigella baumannii 4 strains of OMVs.At present,there is no report on the function of tolA gene in APEC and its influence on the output of OMVs.Therefore,we uses APEC O2 seotype virulent stains E058 as the parent to construct tolA gene deletion strain to study the influence of tolA gene on APEC biological functions and the production of OMVs,and make a preliminary evaluation of the immune protective efficacy of OMVs mediated by it,for providing a reference for the development of vaccines based on OMVs.1 Construction of the tolA-deletion mutant of APEC E058 and evaluation of its biological characteristicsThe tolA deletion mutant was constructed from parental strain E058 by ?-Red homologous recombination technology.The biological characteristics of the wild strain,the tolA-deletion strain and complementary strain were compared.There was no difference in the growth rate of the tolA-deletion mutant and the wid-type strain under the same culuture conditions.Transmission electron microscopy showed that surface of the cell membrane of the tolA-deletion strain was obviously thinner and incomplete.Serum bactericidal test results showed that the anti-bactericidal ability of tolA-deficient strain was significantly reduced at different serum concentrations(0.5%-25%).After the deletion of tolA gene,the motility capacity of bacteria was reduced.The HD11 phagocytosis test showed that the survival rate of tolA-deficient strains in HD11 cells was significantly reduced.The virulence evaluation based on LD50 showed that the virulence of the tolA-deficient strain was reduced by about 100 times compared with the wild-type,while the virulence of the complementary strain reached to the level of the wid-type.The in vivo colonization test of 21-day-old SPF chickens showed that the ability of the deletion strain to colonize in the heart blood,liver and lungs was significantly reduced,indicating that the tolA gene has a significant impact on the virulence of the APEC E058,suggesting that the tolA deletion strain may be used as an attenuated vaccine candidate strains.2 The effects of deletion of tolA gene on the output of Outer Membrane Vesicless in APEC E058The OMVs secreted by the E05 8 and the tolA-deficient strain were extracted by ultrafiltration,concentration and ultra-isolation methods,and the morphology of the OMVs were observed transmission electron microscope.The protein concentration was detected by BCA kit and the protein patterns were analyzed by SDS-PAGE.The effect of OMVs of different concentrations on the activity of related enzymes in HD11 cells was detected.The transmission electron microscope results showed that the OMVs of the wild strain and the tolA-deficient strain had no obvious different in morphology,and they were all spherical double-layer membrane structures.The content of OMVs extracted from the tolA-feficient strain was much higher than that of the wild-type strain.The SDS-PAGE showed that OMVs protein patterns were changed after the deletion of tolA gene.After the co-cuiture of OMVs with HD11 cells.?tolA-OMVs could significantly increase the activities of intracellular acid phosphatase(ACP),lactate dehydrogenase(LDH)and superoxide dismutase(SOD),indicating that tolA-mediated OMVs could activate macrophages and enhance the immune function of macrophages.3 Preliminary study on the immune protective efficacy of OMVs isolated from the tolA-deletion mutant of APEC E058In order to evaluate the immune protection efficiency of OMVs,the extracted OMVs of wild-type and tolA-deficient strain were used to immunize commercial chickens.By extracting bacterial outer membrane proteins(OMPs),an indirect ELISA method was established to detect the level of antibodies produced by OMVs.Inaddition,the overall titer level of immune serum was detected by a glass plate agglutination test.The results showed that OMVs have a good immune protection efficacy,the protection rate of E058-OMVs immunization group can reach more than-80%,and the protection rate of ?tolA-OMVs immunization group can reach more than 90%.As determined by ELISA experiments,the AtolA OMVs immunized group can reach a higher IgG level than that of the E058-OMVs immunized group.The histopathological observation showed that OMVs of the tolA-deficient strain had a obvious inhibitory effect on tissue damage caused by APEC after immunization. |
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