Isolation And Identification Of Riemerella Anatipestifer And Immunological Characteristics Of Recombinant YaeT Protein

Riemerella anatipestifer(RA)is a common bacterial disease in ducks and geese in which incidence rate is over 90%and mortality rate is 5%to 80%.It is a great hazard to the waterfowl industry.In this paper,suspected diseased ducks provided by a farm in Wenchang City,Hainan Province were dissected.The pathogens were isolated and cultured,and then morphologically observation,biochemically detection,PCR amplification,sequencing and drug resistance dection were performed.The anatomical symptoms of diseased ducks were hepatomegaly,pericarditis and pleurisy;The morphology of the bacteria was gram-negative bacilli.Twenty suspected strains were identified by biochemical tests,seventeen of which were identified as RA by PCR and sequencing.The isolated RA was sensitive to ciprofloxacin,amoxicillin,florfenicol,rifampicin,gentamicin,cefoperazone,clindamycin,and ceftizoxime and was strain is resistant to neomycin and Fosfomycin.Outer membrane protein YaeT of RA was analyzed by bioinformatics,including physicochemical properties,homology and structure.The results showed that YaeT is a transmembrane protein composed of ?-helix,random coil and ?-sheet,forming ?-barrel protein.The amino acid sequence homology of YaeT is higher than 97%.It is speculated that the outer membrane protein YaeT is conserved in RA of different serotypes and has the potential to develop a universal vaccine for RA.Genome of RA standard strain ATCC11845 was isolated as a template,and the YaeT gene was cloned by PCR.The prokaryotic expression vector pET28a-YaeT of RA outer membrane protein YaeT was constructed and transformed into E.coli BL21 competent cells for self-induced expression and purification.The results showed that the YaeT gene was highly expressed and purified in E.coli.The duck was immunized with YaeT protein,and immune characteristics of the protein against RA were studied by ELISA,serum bactericidal test,conditioning phagocytosis test and virus challenge test.The antibody titers of ELISA immunized with YaeT protein were 1:583200 and 1:194400 with bacterial ELISA,which were significantly higher than those of PBS group and PBS+adjuvant group.Serum antibodies could promote complement killing pathogens in vitro.The colonies of YaeT protein group were significantly lower than those of PBS group and PBS+adjuvant group(P<0.01),indicating that immune serum could promote phagocytosis of neutrophils.The mortality of PBS group was 60%,PBS+adjuvant group was 50%,and YaeT+adjuvant group was 10%.It showed that the survival rate of ducklings could be greatly improved by inoculating YaeT protein.The results showed that as a genetic engineering vaccine,outer membrane protein YaeT had good immunogenicity and high immune protection rate for RA.It opened up a new research approach for the prevention and treatment of RA,and provided a theoretical basis for the application and promotion of genetic engineering vaccine for RA.