DAO 37:43-52 (1999)  -  doi:10.3354/dao037043

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

Gregory D. Wiens1,*, Maw-Sheng Chien2,**, James R. Winton2, Stephen L. Kaattari3

1Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, Oregon 97201, USA
2Western Fisheries Research Center, 6505 N.E. 65th St, Seattle, Washington 98115, USA
SUP>3Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062, USA
*E-mail:
**Present address: Department of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227, ROC

ABSTRACT: Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salmonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4C11, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a monomer that retains salmonid leucocyte agglutinating activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA, the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer. Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.


KEY WORDS: Bacterial kidney disease · Renibacterium salmoninarum · Monoclonal antibody · Agglutination · Epitope


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