Construction And Bacterial Ghosts Development Of Avian Pathogenic Escherichia Coli AroA-luxS Double Gene Deletion Mutant

Avian pathogenic Escherichia coli (APEC) can cause a variety of diseases which generally known as avian colibacillosis, which resulted in avian acute and chronic infectious diseases. It is a common infectious disease of which mainly characterized with air sacculitis, perihepatitis and pericarditis in the poultry farming industry.At present, the prevention and control of the disease rely mainly on antibiotic treatment and vaccine immunization. But antibiotics abuse problems lead to drug resistance and drug residues, which is a threat to the healthy development of the industry and public health security. And traditional vaccines can only play a role of immune protection against homologous bacteria, there is no or weak crossover protection among serotypes, which makes it hard to prevent and control effectively.Bacterial ghosts is a new technology for vaccine drugs and rendering system. Bacterial ghosts are produced by controlled expression of cloned PhiX174 gene E in Gram-negative bacteria and result in lysis. They are non-living bacterial envelopes, which maintain the cellular morphology and induce the body produce humoral immunity and cellular immunity, so they can be used as a vaccine.In this study, an aroA-deletion APEC mutant (strain DE17△AaroA) and an aroA-luxS double deletion APEC mutant (strain DE17AaroAAluxS) were constructed from the APEC DE17 strain. The immune effect of the bacterial ghosts (BGs) vaccine for prevention and control the avian colibacillosis was evaluated. 1. The epidemiological investigation of APECWe identified 139 APEC isolates from Jiangsu, Anhui and Fujian provinces. The O serotypes of the isolates was identified by agglutination with specific antisera. Virulence-associated genes were investigated by PCR amplification. Drug sensitivity test was carried out according to National Committee for Clinical Laboratory Stands (NCCLS). Agglutination with specific antisera showed that O1, O2 and O78 was the dominant serotypes, which accounted for 28% of the isolates. PCR amplification of the virulence-associated genes showed that iucD, tsh, iss and irp2 were amplified from more than 50% of the strains. Drug sensitivity test showed that 86% of the strains were resistant to more than 10 antibiotics. Fifty per cent of the isolates belonged to the O1, O2and O78 serotypes, which containing five or more virulence genes.The research showed that drug resistance is increasing, we urgently need to adopt new ways to prevent and control avian colibacillosis.2. Construction and characterization of APEC aroA mutant and aroA-luxS doublegene mutantIn this study, an aroA-deletion APEC mutant (strain DE17△aroA) and aroA-luxS double gene deletion APEC mutant (strain DE17△aroA△luxS) were constructed from the DEI7 strain.To evaluate the potential use of DE17△LuxS△aroA as a vaccine candidate,7-day-old ducklings were divided randomly into five groups of ten for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17△luxS, DE17△aroA, and DE17△luxS△aroA were 50.0%,70.0%,70.0, and 80.0% protected.The adherence and invasion results showed that the abilities of DE17△luxS△aroA and DE17△aroA were reduced by 36.5%、 42.5% and 25.8%、 29.3%, as compared to the wild-type strain DE17 (p< 0.01 and 0.05, respectively).Furthermore, in vivo studies showed that the bacterial loads of DE17△lvxS△aroA were reduced by 8400-and 11,333-fold in the spleen and blood of infected ducks, respectively, while those of DE17△aroA were reduced by 743-and 1000-fold, respectively, as compared to the wild-type strain DE17.In addition, the DE17 AaroA and DE17△aroA△luxS showed reduced bacterial motility, compared to wild type strain DE17.The results indicated that double deletion of aroA and luxS genes reduced the bacterial virulence and pathogenicity.3. Development of the bacterial ghosts of DE17△aroA△luxSThe effective lysis plasmid (pUC19-△CI857-E-rrnB-pl-SN), which contained E and SN genes was constructed.The bacteriolytic results showed that the new lysis plasmid has stably repressed the expression of gene E at temperatures of up to 37 ℃, and the lysis efficiency of APEC reached to 99.99%. The bacterial ghosts showed extent cytoplasm discharges by TEM observation.For ensure the safety of BGs vaccine, SN was sended into the lysis plasmid which expression could cause bacterial genome degradation and improve inactivation of the bacteria. Then treatment of freeze-dry or addition of 40 μg/mL gentamicin ensured the 100% of lysis efficiency of the bacterial ghosts.4. Protection evaluation of the the bacterial ghostsSeven-day-old ducklings were immunized and challenged with 20 LD50 of DE17 on day 14 post immunization. The protection rate was 50% for the ducklings immunized with formaldehyde inactivated DE17 with adjuvant. However, the protection rate reached to 70%for the formaldehyde inactivated DE17△aroA and 80% for the double gene mutant DE17△aroA△luxS. The bacterial ghosts induced better humoral immunity than formaldehyde inactivated DE17, and generated high level IgG than formaldehyde inactivated DE17.In this study, the mutants of DE17△aorA and DE17△aor△dluxS were constructed from DE17, and an efficient BGs based on the lysis plasmid was prepared. The present results showed that BGs vaccine induced better humoral immunity and provided higher protection than traditional inactivated vaccine,which indicated that the bacterial ghost could be developed as an ideal vaccine candidate in the future.