Characterization of a Major Outer-Membrane Antigen ofEdwardsiella ictaluri

Abstract

Edwardsiella ictaluri is the cause of enteric septicemia of catfish. A monoclonal antibody (MAb AA224) was used to identify a specific and predominant outer-membrane antigen of E. ictaluri. The MAb AA224 was produced by conventional cell fusion technology with spleen cells from mice immunized with an affinity-purified antigen. The affinity-purified antigen was obtained by immunoaffinity chromatography of an E. ictaluri extract with immunoaffinity purified immunoglobin from sera of channel catfish let alums punctatus immune to E. ictaluri as a result of natural infection. The imrnunoaffinity-purified antigen was used for immunization and identification of the hybridoma producing MAb AA224 by enzyme-linked immunosorbent assay. The predominant antigen was purified by immunoaffinity chromatography with MAb AA224 as the immunoadsorbent. Immunoblotting and high-pressure liquid chromatography were used to determine that the relative sizes of the predominant antigens are 60 and 36 kilodaltons. Immunoelectron microscopy with MAb AA224 conjugated with colloidal gold localized the predominant antigen on the surface of the outer membrane of £. ictaluri.

Immune response induced by N-lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish

“…Several studies have recognised the importance of OMPs for virulence of E. ictaluri and characterised the proteins for a number of isolates [16,17,19], but none have characterised the AL-93-75 isolate used in this study. Newton et al [16] first characterised OMPs from isolate ATCC 33202 and 32 other isolates on SDS-PAGE.…”

“…Lipopolysaccharide and crude cell wall extracts from E. ictaluri (AL-93-75) were prepared according to Klesius et al [17].…”

“…The primary antibody was sera from channel catfish vaccinated with the OMP fraction used at a 1:200 dilution. The secondary antibody was goat anti-channel catfish Ig heavy chain [17] used at a 1:2000 dilution. The tertiary antibody was alkaline-phosphatase conjugated anti-goat IgG (Sigma, St Louis, MO, USA) used at a 1:2000 dilution.…”

“…These OMPs have been shown to contain several important bacterial virulence factors including fimbriae, and porins [14,15]. Also, less purified OMP fractions have already been shown to be immunogenic in channel catfish [16][17][18][19]. This study is the first to use AL-93-75, a virulent isolate of E. ictaluri as the source of a highly purified OMP fraction.…”

Cloning and characterization of Edwardsiella ictaluri proteins expressed and recognized by the channel catfish Ictalurus punctatus immune response during infection

“…Catfish that survive an Edwardsiella ictaluri infection are generally assumed to be resistant to ESC when they recover (Klesius & Horst 1991). The bacterium is considered a good candidate for vaccine development because it has a very high serologic homogeneity (Plumb 1988, Plumb & Vinitnantharat 1989, Bertolini et al 1990), although reported efficacy using killed E. ictaluri bacterins has been equivocal at best , Saeed & Plumb 1986, Thune et al 1993, 1997b.…”