Progress 07/01/03 to 06/30/05
Outputs
This project focused on the role of major virulence protein, p57, in binding and attachment to fish leukocytes. We utilized several approaches to map the p57 leukocyte-binding domain. In our first approach, we mapped the epitopes recognized by three neutralizing monoclonal antibodies using transposon mutagenesis and synthetic peptides. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between positions 78 and 210 disrupted the 4C11 epitope, and insertions between 158 and 234 disrupted the 4D3 epitope. The three monoclonal antibodies failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. From these data, we conclude that the binding domain is located in the amino-terminus of p57 and that recognition of secondary structure is important for neutralization. In our second approach, we mapped the presence or absence of the 4D3, 4C11 and 4H8 epitopes on
clinical isolates of R. salmoninarum. We screened twenty-nine isolates from diverse geographic locations and identified eight isolates lacking the 4C11 epitope. These antigenic variants originated from either Norway or British Columbia. We determined the molecular basis of the loss of the 4C11 epitope in strain 684 and determined that both copies of the msa gene, encoding p57, contain a single Ala139 to Glu substitution. P57 purified from strain 684 displayed enhanced leukocyte-binding and agglutinating activity. These data are in agreement with the transposon mutagenesis results demonstrating the importance of the p57 amino-terminus for leukocyte-binding and were the first direct evidence of antigenic variation in p57. A manuscript describing the extent of antigenic variation in a large number of strains and the results from attachment/invasions assays is in preparation. A final accomplishment of this project was the determination of genome size of Renibacterium salmoninarum by
pulsed-field gel electrophoresis. The genome was estimated to be 3.4 + .1 Mb and six genes were mapped to restriction fragments.
Impacts
Improved diagnostic assays and a specific vaccine against bacterial kidney disease are needed by the aquaculture industry and fish health managers. In this project, we characterized monoclonal antibodies that neutralize an important virulence factor, p57. This led to the identification of several antibodies that can be used to improve the sensitivity and specificity of a commercial monoclonal antibody-based ELISA. This work also resulted in the identification of neutralizing determinants on p57 that could be used in a vaccine. Project funds were used to determine the genome size of Renibacterium salmoninarum. This data was critical for acquiring funding for a new multi-state project between The National Center for Cool and Cold Water Aquaculture, Kearneysville WV, National Marine Fisheries Service, NOAA, Seattle, WA, Oregon State University, Corvallis, OR and Integrated Genomics, Chicago IL to completely sequence the genome of Renibacterium salmoninarum.
Publications
- Wiens, G.D., and Owen, J. 2005. Mapping neutralizing epitopes on Renibacterium salmoninarum p57 using transposon mutagenesis and synthetic peptides. Applied and Environmental Microbiology. 71:2894-2901
- Wiens, GD. 2005 Bacterial Kidney Disease. In: Aquaculture Compendium. Wallingford, UK: CAB International. In Press.
- Wiens GD, Strom MS. 2005. Estimation of the Genome Size of Renibacterium salmoninarum ATCC 33209 by Pulsed-Field Gel Electrophoresis. Abstract 30th Eastern Fish Health Workshop, Shepherdstown, WV. Page 8.
|
Progress 10/01/03 to 09/30/04
Outputs
During the reporting period, this project was transferred from Oregon Health and Sciences University to the National Center for Cool and Cold Water Aquaculture, WV. The following progress was made. We completed transposon mutagenesis and peptide mapping to identify neutralizing epitopes on p57. The analysis of p57 antigenic variation was extended and additional isolates containing a single Ala 139 to Glu substitution were characterized. P57 deletion mutants were constructed. The genome size of Renibacterium salmoninarum was determined by pulsed-field gel electrophoresis.
Impacts
Bacterial kidney disease is a prevalent disease that impacts the production of farmed salmon and trout. Improved diagnostic assays and a specific vaccine are needed by the aquaculture industry and fish health managers. During this reporting period, we have characterized monoclonal antibodies that neutralize an important virulence factor, p57. This has led to the identification of several antibodies that can be used to improve the sensitivity and specificity of a commercial, monoclonal antibody ELISA. We also identified neutralizing determinants on p57 that may be used for vaccine development. Project funds were used to determine the genome size of Renibacterium salmoninarum. This data was critical for acquiring funding for a new multi-state project between the National Marine Fisheries Service, (Seattle, WA), the National Center for Cool and Cold Water Aquaculture (Kearneysville, WV), Oregon State University (Corvallis, OR) and Integrated Genomics (Chicago, IL) to
completely sequence the genome of Renibacterium salmoninarum.
Publications
- Wiens, G.D., and Owen, J. 2005. Mapping neutralizing epitopes on Renibacterium salmoninarum p57 using transposon mutagenesis and synthetic peptides. Applied and Environmental Microbiology. In Press
|
|