title: Development of an ELISA using a recombinant 41 kDa partial protein (P45N') for the detection of Riemerella anatipestifer infections in ducks.
abstract: Riemerella anatipestifer, a gram-negative bacillus, is the causative agent of duck septicemia, a disease which could incur much economic loss in the duck industry. An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to facilitate early detection of R. anatipestifer infection in ducks. The antigen used was a recombinant 41 kDa N-terminal fragment (rP45N') of a newly characterized R. anatipestifer potential surface protein, P45, which was expressed in Escherichia coli as an N-terminal GST fusion protein. The rP45N'-based ELISA successfully detected P45 antibodies in the sera of 20 ducks immunized with bacterin preparations of R. anatipestifer serotypes 1, 10 15, 19 and the ATCC11845 strain. Antibodies to P45 were also detected in the sera of 25% (75/296) of White Pekin ducks which were imported into Singapore from three different farms. Successful discrimination was obtained between sera from infected ducks and that of specific-pathogen free ducks (p<0.01). The rP45N'-GST antigen did not cross-react with antibodies in sera from guinea pigs which were infected with other gram-negative and gram-positive bacterial pathogens, including Aeromonas hydrophila, Citrobacter freundii, E. coli, Klebsiella pneumoniae, Pastuerella multocida, Proteus mirabilis, Salmonella spp., Serratia maccescens, Shigella sonnei and Yersinia enterocolitica. In addition, the DNA sequence encoding P45 was detected in R. anatipestifer serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 19 and the ATCC11845 strain, suggesting that P45 is probably also universally expressed in these R. anatipestifer serotypes. Thus, the ELISA described is applicable to the detection of R. anatipestifer infection in ducks.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Institute of Molecular Agrobiology, National University of Singapore, 117604, Singapore, Singapore.
authors.forename: Bin
authors.initials: B
authors.lastname: Huang
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
authors.forename: Hilda
authors.initials: H
authors.lastname: Loh
authors.forename: Joachim
authors.initials: J
authors.lastname: Frey
authors.forename: Hai-Meng
authors.initials: HM
authors.lastname: Tan
authors.forename: Kim-Lee
authors.initials: KL
authors.lastname: Chua
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001425:Bacterial Outer Membrane Proteins; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000235:genetics / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011201:Poultry Diseases / Q000175:diagnosis / Q000382:microbiology*; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary; D017422:Sequence Analysis, DNA; D013047:Specific Pathogen-Free Organisms
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins