Construction Of LpxLLpxMLpxP Mutants Of Shiga Toxin-producing Escherichia Coli O141and O8Causing Post-weaning Diarrhea And Edema Disease Of Pigs And Evaluation Of Their Immune Efficacy

Porcine post-weaning diarrhea and edema disease caused by Shiga toxin-producing Escherichia coli (STEC) is one of the most common infectious diseases in pigs, and is often infected with combination of other pathogens such as other bacteria or viruses. It is responsible for worldwide economic losses to the pig industry.As a Gram-negative bacterium, the STEC cell envelope includes two lipidbilayers, an inner membrane composed primarily of phospholipids, and an outer membrane containing primarily phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The LPS molecule can be divided into three parts:lipid A, core polysaccharides and O-antigen repeats. Tight stacking of the long-chain fatty acids of lipid A, the hydrophobic anchor of LPS, forms a permeability barrier around the cell. Lipid A plays an important role in the pathogenesis of Gram-negative bacterial infections. It potently activates the innate immune system, triggering the synthesis of cationic antimicrobial peptides, cytokines, clotting factors, and other immunostimulatory molecules. The lipid A contains four "primary"(R)-3-hydroxyacyl chains. Some of the (R)-3-hydroxy groups are further modified with "secondary" acyl chains, forming acyloxyacyl moieties. In Escherichia coli, the final steps of lipid A synthesis occur in the inner membrane by HtrB (LpxL) and MsbB (LpxM). An additional ACP-dependent late acyltransferase, LpxP, is induced upon cold shock (12℃). LpxP incorporates the unsaturated fatty acid palmitoleate in the place of laurate. In this study, genes in STEC serogroup0141and08strains chosen for deletion were LpxL, LpxM and LpxP. The knockout mutants were named as O141ΔLpxLALpxMALpxP and O8ALpxLALpxMALpxP. Southern blot showed single copy of FRT gene was inserted into the chromosome DNA of STEC0141strain and08strain;RT-PCR showed that the insertion didn’t have a polar effect on the upstream or downstream genes trascription of LpxL, LpxM or LpxP. Compared with their parental strain0141and08, LpxLLpxMLpxP mutants had a reduced growth rate in LB broth. These results indicated that the LpxM, LpxL and LpxP genes were important for the growth of STECThe5-week-old mice were immunized with the inactivated vaccine of recombinant starins0141ΔLpxLΔLpxMALpxP and OSΔLpxLΔLpxMALpxP by the peritoneal route. Sera and feces were simultaneously collected from the vaccinated mice on the7,14days post first immunization and on the1,14days post second immunization. The result indicated that serum IgG specific to F18and Stx2e antigens could be stimulated in mice immunized with the inactivated vaccine, but not in all oral groups. Titers of IgG from sera against F18and Stx2e antigens were measured by indirect ELISA respectively.The protection efficacy of vaccinated mice against challenge of strain0141and08were above80%.