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United States Department of Agriculture

Agricultural Research Service


Location: Cool and Cold Water Aquaculture Research

2005 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
Efficient aquatic animal production is limited by the occurrence of endemic and emerging diseases. It is estimated that disease-related losses in US finfish aquaculture approach $200 million annually. In the rainbow trout industry, about 30% of production is lost to infectious disease. In addition to direct loss, disease also increases production costs due to reduced growth and food conversion, and increased need for intervention. The lack of adequate technology for disease prevention and treatment, have hindered industry growth and competitiveness. Currently, only two antibiotics and a few chemicals are approved for use with food fish. Their use has led to concerns about antibiotic resistance and the impact of these chemicals on the environment. This project focuses on two significant bacterial pathogens of trout are Yersinia ruckeri, the causative agent of enteric redmouth disease; and Flavobacterium psychrophilum, the causative agent of bacterial cold water disease. Most rainbow trout are vaccinated against enteric redmouth disease; however, the use of the standard immersion vaccine is not always effective and the mechanisms of protective immunity are unknown. Control of bacterial cold water disease is a major priority of the trout industry as there is no commercial vaccine against F. psychrophilum. There are three specific goals of this project:.
1)characterize host-pathogen interactions leading to the disease state and develop highly effective tools for pathogen detection and control,.
2)define mechanisms controlling innate and acquired immunity to aid in both vaccine design and development of disease resistant strains of rainbow trout, and.
3)develop novel engineering-based strategies that improve fish health in intensive fish culture environments. This research is intended to increase production efficiency for fish farmers of cool and cold water aquaculture species, and provide scientists and government agencies that manage farmed and wild fish stocks, information on host-pathogen interactions.

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.

2.List the milestones (indicators of progress) from your Project Plan.
The major objectives of the project are: 1. Characterize virulence determinants produced by the rainbow trout pathogens Yersinia ruckeri and Flavobacterium psychrophilum. 2. Identify rainbow trout genes and factors that are critical to expression of innate and acquired immunity against Y. ruckeri and F. psychrophilum. 3. Identify water quality factors that influence host susceptibility and pathogen refuge within a recirculating system, and evaluate the use of bacteriophage as a targeted antibacterial strategy in recirculating and serial reuse aquaculture systems. Milestones listed by year: Year 1 (FY 2005) 1. Development of a green fluorescent strain of Y. ruckeri for disease challenge studies. 2. Complete 8x shotgun sequence coverage of the F. psychrophilum genome, cosmid library construction and automated annotation using ERGO. 3. Complete sequencing of 54 rainbow trout cytokine cDNA clones. 4. Evaluate bacterial cold water disease resistance in 75 full-sib NCCCWA family fish from the 2005 brood year. 5. Collect samples and measured steroid hormone accumulation in a production-scale recirculating aquaculture system. 6. Complete genome sequencing of Y. ruckeri-specific phage NC10.

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.

4a.What was the single most significant accomplishment this past year?
Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a serious problem for rainbow trout aquaculture. Conventional approaches to vaccines have been ineffective and antibiotic resistance is of concern. To address this major health problem, two complementary programs at the NCCCWA are underway. These programs are to.
1)determine the complete bacterial genome sequence in order to identify targets to develop an effective vaccine, and.
2)selectively breed rainbow trout for increased disease resistance. This year, in coordination with two other CRIS projects at the Center, we evaluated 75 full-sib families for resistance to Flavobacterium psychrophilum intraperitoneal injection challenge. A large variability in disease resistance was observed between families ranging from highly susceptible (1% survival) to highly resistant (72% survival). Tissue samples were collected from 4800 fish for DNA extraction and identification of genes involved in disease resistance. This study identified rainbow trout crosses used for further propagation at the NCCCWA.

4b.List other significant accomplishments, if any.
1. Creation of a green fluorescent protein-tagged strain of Yersinia ruckeri- An engineered strain of Yersinia ruckeri has been developed which constitutively expresses the green fluorescent protein (GFP). GFP expression is exceptionally stable and the strain retains it ability to colonize and cause disease in immersion-challenged rainbow trout. This strain has been used for detecting and imaging the interactions between this pathogen and its natural salmonid host. This strain is also easily identified in complex environmental samples making it useful for characterizing factors that allow its persistence and spread outside its host.

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.

4c.List any significant activities that support special target populations.

4d.Progress report.
1930-32000-002-01R: This report serves to document research conducted under a reimbursable agreement between the ARS and the USDA, CSREES National Research Initiative Competitive Grants Program. Project progress includes the identification of additional antigenic variation in p57 and testing two vaccine candidates derived from genome sequencing.

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.

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
Project started 1/11/05.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Wiens, GD. 2005 Fish Health Research at the National Center for Cool and Cold Water Aquaculture. Fish Tales Newsletter. West Virginia University Extension Service. Fall Newsletter.

Review Publications
Welch, T.J., Crosa, J. 2005. Novel role of the lipopolysaccharide o1-side chain in ferric-siderophore transport and virulence of vibrio anguillarum. Infection and Immunity Vol. 73 No 9, 5864-5872.

Wiens, G.D., Owens, J. 2005. Mapping neutralizing epitopes on renibacterium salmoninarum p57 using transposon mutagenesis and synthetic peptides. Applied and Environmental Microbiology 71 (6): 2894-2901.

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).

Last Modified: 7/24/2014
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