Little is known about the virulence factors of Renibacterium
salmoninarum, the causative agent of salmonid bacterial kidney disease.
The predominant protein produced by R. salmoninarum in broth culture or
during infection is a 57/58 kDa protein (p57) which is associated with strain
virulence. In this study monoclonal antibodies (MAbs) to p57 were developed
and used as tools to antigenically characterize and quantify the protein.
Monoclonal antibodies 4D3 and 2G5 recognize p57 and appear to be species
specific as they did not cross-react with proteins produced by bacterial
species within the genera Streptococcus, Carnobacterium, Vibrio and
Aeromonas, or with fish serum proteins. Further, these MAbs recognize
conserved epitopes on p57 shared by 10 isolates from geographically diverse
areas.
In vitro activities attributed to p57 include the suppression of antibody
production, and the agglutination of rabbit erythrocytes and salmonid
spermatocytes. We described a novel in vitro agglutinating activity of p57
toward salmonid leukocytes that was inhibited by two of a panel of eight
MAbs. The location of the putative epitopes recognized by the MAbs were
determined by two-dimensional electrophoresis and Western blotting of
proteolytic breakdown fragments of p57. Amino acid sequencing of several of
the fragments suggested that the antibodies which inhibit agglutinating activity
bind proximal to the amino terminus of the protein.
To investigate the mechanism of leukocyte agglutination, p57 was
purified to near homogeneity using anion-exchange and size-exclusion fastpressure
liquid chromatography. P57 eluted as a protein monomer and
retained leukoagglutinating activity. In addition, results of antibody-capture,
enzyme-linked immunosorbent assays suggest that a monomer exists in
culture supernatant and infected fish tissue.
Antigenic analysis with MAbs has also been useful for developing
immunoassays for detecting and quantifying p57 levels in vivo. Using a
quantitative ELISA, the prevalence of salmon with antigen levels above 3
ng/ml of kidney homogenate varied from 12.8 to 36.6% in 740 adult spawning
chinook salmon returning to an Oregon hatchery from 1989 to 1991. A rapid,
semi-quantitative, Field ELISA was also developed for use under hatchery
conditions, in addition to a sensitive chemiluminescent Western blot protocol
for confirming ELISA positive samples. / Graduation date: 1992
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36722 |
| Date | 07 February 1992 |
| Creators | Wiens, Gregory D. |
| Contributors | Kaattari, Stephen L. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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