2005 Annual Report
The research in this project falls under National Program 106 - Aquaculture and addresses goals in health management, aquaculture production systems and sustainability and environmental compatibility of aquaculture, as described in the National Program Action Plan.
Year 2 (FY 2006) 1. Construct a bank of Y. ruckeri transposon mutants, design and validate mutant screening procedures. 2. Complete gap closure and manual annotation of the F. psychrophilum genome and deposit annotated genome sequence in GenBank. Identify and evaluate potential vaccine candidates. 3. Develop assays to measure trout cytokine mRNA, determine tissue expression, and develop a rainbow trout immune-gene database. 4. Construct two research-scale recirculating aquaculture systems to determine how water replacement rate alters the composition and quantity of dissolved organic constituents. 5. Complete phage NC10 genome annotation and submission to GenBank. Construct biofiltration systems for use in phage control experiments.
Year 3 (FY 2007) 1. Begin mutant screening and identify ten attenuated Y. ruckeri mutants. 2. Publish F. psychrophilum genome sequence and evaluate vaccine candidates. 3. Construct a rainbow trout immune gene microarray. 4. Evaluate bacterial cold water disease resistance in 75 full-sib NCCCWA fish crosses from the 2007 brood year. 5. Determine whether changes in dissolved organic compounds, resulting from different water replacement rates, alter rainbow trout hematological and physiological parameters. 6. Formulate and validate models for phage biocontrol experiments, and assess the effect of phage treatment on biofilter performance.
Year 4 (FY 2008) 1. Identify genes responsible for Y. ruckeri attenuation. 2. Evaluate F. psychrophilum vaccine candidates. 3. Complete RNA extraction from vaccinated and challenged NCCCWA rainbow trout and perform microarray hybridization experiments. 4. Determine whether extended exposure to highly recycled water containing elevated organic loads changes rainbow trout disease susceptibility. 5. Assess the potential use of phage NC10 for Y. ruckeri biocontrol.
Year 5 (FY 2009) 1. Test two Y. ruckeri prototype live-attenuated vaccines. 2. Evaluate dose, duration and efficacy of lead F. psychrophilum vaccine candidate and identify CRADA partner. 3. Analyze global immune gene expression and identify factors correlated with protective immunity. 4. Evaluate F. psychrophilum disease resistance in 75 full-sib NCCCWA fish crosses, 2009 brood year. 5. Determine if the distribution and number of GFP-tagged Y. ruckeri increase in RAS operated using low water replacement rates. 6. Complete assessment of the utility of using bacteriophage as a Y. ruckeri biocontrol agent.
2. Rainbow Trout Immune Gene Identification Cytokines are critical mediators of innate and acquired immunity; however, few have been identified in fish. Last year we identified a number of putative cytokine sequences in the Institute for Genomic Research (TIGR) rainbow trout, expressed sequence tag (EST) database. This year we completed sequencing of fifty-four unique cDNA clones and have analyzed predicted protein sequences.
3. Continued progress on F. psychrophilum genome sequencing In order to develop an effective vaccine, detailed molecular information of the pathogen is needed. We have continued research initiated last year to completely sequence the genome of Flavobacterium psychrophilum CSF-295-93 in collaboration with Clear Springs Foods Inc. Eight-fold genome sequence coverage has been completed in conjunction with Integrated Genomics. A total of 40,527 shotgun sequencing reads and 2966 fosmid sequence reads were determined and assembled. A total of 2654 open reading frames have been identified and half of these genes were annotated using ERGO bioinformatics software.
4. Analysis of secreted hormones in intensive recirculating aquaculture systems. In order to conserve water, intensively managed recirculating aquaculture systems are operated at low freshwater makeup rates. Under such water regimes, periodic fish health problems arise which have been attributed, but not specifically linked, to dissolved organic compounds that accumulate in the culture water. To determine specific compounds believed to impact fish health during intensive production, four operating conditions were examined and water samples collected to identify direct correlations between 3 steroid hormone compounds known to be excreted by rainbow trout. Cortisol measurements were completed and the ambient concentrations were significantly correlated with freshwater makeup rate.
5. Genomic sequencing of Phage NC10- Last year we identified a bacteriophage that specifically infects and kills the bacterial trout pathogen Yersinia ruckeri. This year we completed sequencing of the phage genome. Shotgun sequencing of a small-insert library yielded 788 sequences that assembled into a linear genome of approximately 69.6 kilobase pairs. Future work will focus on analyzing this sequence to determine the risks of using this bacteriophage as an antibacterial agent.
1930-32000-002-02R: This report serves to document research conducted under a reimbursable agreement between ARS and the Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, Seattle, WA. Project progress includes completion of shotgun sequencing, automated gene identification and annotation.
1930-32000-002-03N: This report serves to document research conducted under a non-funded cooperative agreement between ARS and Clear Springs Foods, Inc. Eight-fold genome sequence coverage has been completed in conjunction with Integrated Genomics. A total of 40,527 shotgun sequencing reads and 2966 fosmid sequence reads were determined and assembled. A total of 2654 open reading frames have been identified and half of these genes were annotated using ERGO bioinformatics software.
Wiens, G.D., Strom, M.S. 2005. Estimation of the genome size of renibacterium salmoniarum atcc 33209 by pulsed-tield gel electrophoresis. (Abstract). 30th Eastern Fish Health Workshop. p. 8.
Welch, T.J., Wiens, G.D. 2005. Construction of a virulent, green fluorescent protein-tagged yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge. Diseases of Aquatic Organisms. 67:267-272.
Wiens, G.D., Gahr, S.A., Morrison, C., Palti, Y., Rexroad III, C.E., Rodriguez, M.F., Welch, T.J. 2004. Genomic characterization and expression analysis of a tumor necrosis factor superfamily 13b (tnfsf 13b) homologue from rainbow trout (oncorhynchus mykiss). International Immunology Congress p. 82C (abstract W15.15).
Strom, M., Wiens, G.D., Rockey, D. 2004. Genome sequencing of the vertically-transmitted fish pathogen renibacterium salmoninarum. Meeting Abstract P 90-93.
Cain, K., Sudheesh, P.S., Lapatra, S.E., Wiens, G.D., Lafrentz, B.R., Call, D.R. 2005. Identification and expression of an immuno-reactive heat shock protein from flavobacterium psychrophilum. Annual Meeting of the Fish Health Section/American Fisheries Society. (abstract).