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United States Department of Agriculture

Agricultural Research Service


Location: Cool and Cold Water Aquaculture Research

2006 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? Why 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 raises production costs due to.
1)slower growth,.
2)decreased food conversion, and.
3)increased need for intervention. The lack of adequate technology for disease prevention and treatment have hindered industry growth and competitiveness. Currently, only a few antibiotics and 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: 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, immersion 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 efficiencies 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 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.

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 by year the currently approved milestones (indicators of research progress)
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 measure 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.List the single most significant research accomplishment during FY 2006.
New Trout Immune Genes Identified. A better understanding of the immune system is required to improve health and disease resistance of cultured fish. This year we published the discovery of two new rainbow trout chemokine genes: CXCd1 and CXCd2. These genes were expressed in most tissues of healthy rainbow trout. Levels of gene expression increased in the major immune organs (spleen and anterior kidney) following either vaccination with dead bacteria or infection with the live Yersinia ruckeri bacteria. Infection of trout with another pathogen, the infectious hematopoietic necrosis virus, did not increase levels of gene expression indicating that the CXCd genes are not up-regulated in response to all pathogens. Our findings suggest that measurement of CXCd1 and CXCd2 gene expression may be of future interest as biomarkers for an ongoing immune response to Yersinia ruckeri. This work furthers the understanding of the fish inflammatory response and how vaccines elicit protective immunity. This accomplishment addresses research outlined in the National Program 106 Action Plan (Integrated Aquatic Animal Health Management: Immunology and Disease Resistance Goals 1 and 2).

4b.List other significant research accomplishment(s), if any.

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

4d.Progress report.
Installation of Recirculating Aquaculture Systems. The NCCCWA in conjunction with the Conservation Fund’s, Freshwater Water Institute completed construction of six recirculating aquaculture systems. These intensive fish culture systems employ biological and mechanical filtration to remove ammonia and suspended solids to maintain water quality conditions that promote fish health and growth. These systems will allow evaluation of the impact of the different operational/management strategies on water quality and fish production in replicated systems over the next three years of this project. These systems will be used to improve design and performance criteria in order to increase rainbow trout production efficiency and sustainability. Additionally, the systems will facilitate a better understanding of the interactions between fish physiology and water quality. This research addresses several components in National Program 106 Action Plan (Sustainability and Environmental Compatibility: Water Use and Reuse Goals 1 and 2; and Aquatic Animal Health Management Goal 2).

1930-32000-002-03N: This report serves to document research conducted under a Non-Funded Cooperative Agreement between ARS and Clear Springs Foods, Inc. 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. This year, the remaining gaps in the F. psychrophilum genome sequence were determined and the results indicate that the genome is composed of a single, circular chromosome. We have completed the preliminary identification and annotation of genes in the DNA sequence. The identified genes are the basis of ongoing vaccine studies scheduled to be completed over the next three years. This report serves to document research conducted under a Non-Funded Cooperative Agreement between ARS and Clear Springs Foods Inc. This work also addresses research outlined in the National Program 106 Action Plan (Integrated Aquatic Animal Health: Microbial Genomics Goal 1).

Selection of Trout with Increased Resistant to Bacterial Cold Water Disease. Last year, in coordination with two other CRIS projects at the NCCCWA, we evaluated the innate resistance of 75 full-sib families to injection challenge with F. psychrophilum. Fish averaged 2.4 g in size and a large variability in disease resistance was observed between families ranging from highly susceptible (1% survival) to highly resistant (72% survival). This year, we repeated the challenge on 4 susceptible and 4 resistant families from the 2005 brood year when the fish had grown to an average of 10 g and 800 g in size. Six of eight families maintained their original resistance/susceptibility phenotype suggesting that this trait is manifested throughout the trout rearing cycle. The mechanism of resistance is under investigation. This research contributes to the development of disease resistant rainbow trout and addresses objectives outlined in the National Program 106 Action Plan (Integrated Aquatic Animal Health Management: Immunology and Disease Resistance Goal 4).

1930-32000-002-01R: This report serves to document research conducted under a Reimbursable Agreement between the ARS and the USDA National Research Initiative Competitive Grants Program and was completed during this reporting period. Additional details of research can be found in the report for the parent CRIS 1930-3200-002-00D Host, Pathogen and Environmental Interactions in Cool and Cold Water Aquaculture. This project focused on the role of major virulence protein, p57, in binding and attachment to fish leukocytes. Several approaches were utilized to map the p57 leukocyte-binding domain and to investigate antigenic variation in p57. Epitopes recognized by three neutralizing monoclonal antibodies were mapped using transposon mutagenesis and synthetic peptides. From these data, it was determined that the binding domain is located in the amino-terminus of p57 and that recognition of secondary structure is important for neutralization. Antigenic variation was examined in a panel of 24 R. salmoninarum isolates obtained from diverse geographic locations world-wide using six p57-specific monoclonal antibodies (Mabs). Full-length p57 was produced by all 24 isolates and for each isolate, the protein was associated with the bacterial cell surface. The epitopes were recognized by four Mabs (4D3, 4H8, 3H1 and 1A1) which were conserved among all strains tested. The 4D10 epitope was disrupted in one isolate from British Columbia, while the 4C11 epitope was lost in 5 of 8 strains isolated from Norway. These results suggest that while p57 is not highly polymorphic, some antigenic variation exists that may be geographically restricted in distribution. This research led to the identification of several antibodies that can be used to improve the sensitivity and specificity of a commercial, monoclonal antibody-based ELISA used to identify infected salmon and trout. This work also resulted in the identification of neutralizing determinants on p57 that could be used in a vaccine.

1930-32000-002-02R: This report serves to document research conducted under a Reimbursable Agreement between the ARS and the Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, Seattle, WA. Additional details of research can be found in the report for the parent CRIS 1930-3200-002-00D Host, Pathogen and Environmental Interactions in Cool and Cold Water Aquaculture. The project title is “Genome sequencing of the vertically-transmitted fish pathogen Renibacterium salmoninarum”. The following progress was made during the reporting period. The genome was closed in October 2005 by the University of Washington Genome Center. 9 X sequence coverage was obtained from small-insert library sequencing and the genome assembled using PHRED/PHRAP/CONSED software tools. The genome was closed by four rounds of AUTOFINISH followed by manual assembly. A total of 52,686 sequence reads were incorporated in the final assembly and the gross assembly was validated by pulsed-field gel electrophoresis. The genome of R. salmoninarum is a single, circular chromosome of 3,155,250 base pairs with 56.27% G+C content. A total of 3667 open reading frames were identified by Integrated Genomics ORF-Calling software. Using the program PSORTb v2.0, ORFs were classified as putative cytoplasmic (n=1626), cytoplasmic membrane (n=625), cell wall (n=16), extracellular (n=132), or unknown localization (n=1268). We have combined these data with additional motif searches and immunogenicity profiles to develop a list of potential vaccine candidates for further examination. Additionally, trout immune gene expression in response to R. salmoninarum ATCC 33209 infection was measured. A workshop titled “Bacterial Kidney Disease: Challenges for the 21st Century,” sponsored in part by project funds and organized by the PIs, was held in Seattle WA November 15-17, 2005. At this international workshop (, one day was devoted to the announcement of first findings obtained through the R. salmoninarum Genome Project. Knowledge of the R. salmoninarum genome will serve as a foundation for identification of virulence factors, vaccine gene candidates and improved diagnostic tests. In addition, the completed R. salmoninarum genome sequence will be a template for comparison with clinical and environmental isolates.

5.Describe the major accomplishments to date and their predicted or actual impact.
Development of a Disease Challenge Model for Assessing Immunity and Bacteriophage Therapy. Standardized disease challenge models are necessary to understand immune mechanisms and for the advancement of novel therapeutics. Last year, we developed an engineered strain of Yersinia ruckeri which constitutively expresses the green fluorescent protein (GFP) and retains its ability to colonize and cause disease in immersion-challenged rainbow trout. The expression of GFP allowed the rapid imaging of the pathogen in host tissues and the interaction with immune cells. After bath or intra-peritoneal challenge, intracellular bacteria were detected in peripheral blood, spleen and anterior kidney leukocytes by flow cytometry and fluorescence microscopy. This system will be useful for understanding of the routes of infection, the host cellular response, and immune gene regulation in response to vaccination.

This model has also been useful for evaluating bacteriophage as novel antimicrobials. A lytic bacteriophage (Phage NC10) which lyses Y. ruckeri was identified from sediments collected at a trout farm. The phage genome as been sequenced and no virulence factors were present. Current investigations are focused on assessing the effectiveness of phage YRNC10 for the treatment of Y. ruckeri infections and for the reduction of pathogen loads in aquaculture systems. Bacteriophages have potential as highly specific and self-replicating, natural, antimicrobials.

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. Trout Talk. Fall Newsletter.

Review Publications
Wiens, G.D., Glenney, G.W., Lapatra, S.E., Welch, T.J. 2006. Identification of novel rainbow trout (Onchorynchus mykiss) chemokines, cxcd1 and cxcd2: Mrna expression following Yersinia ruckeri vaccination and challenge. Immunogenetics. 2006 May;58(4):308-23. Epub 2006 Mar 29.

Glenney, G., Wiens, G.D. 2006. Early diversification of the tumor necrosis superfamily in teleosts: genomic characterization and expression analysis. 10th International Congress, International Society of Developmental and Comparative Immunology. Meeting Abstract Page 113.

Wiens, G.D. 2005. Novel vaccine targets and assessment of cellular immunity.Workshop on genomics of Renibacterium saloninarum. Meeting Abstract Bacterial Kidney Disease: Challenges for the 21st Century. p. 12.

Wiens, G.D., Ole Bendik, D. 2005. Antigenic variation in Renibacterium salmoninarum p57: functional and diagnostic implications. Bacterial Kidney Disease: Challenges for the 21st Century. Meeting Abstract. November 15-17, 05 Seattle, WA p. 21.

Welch, T.J. 2005. Characterizaion of a bacteriophage specific for the salmonid pathogen yersinia ruckeri. Evergreen International Phage Meeting. the Evergreen State College, Olympia, WA. p. A-43.

Brazil, B.L., Silverstein, J. 2005. Growth and physiological characteristics of rainbow trout reared in different culture environments. Meeting Abstract, Volume 12.

Brazil, B.L. 2006. Performance and operation of a rotating biological contactor in a tilapia recirculating aquaculture system. Aquacultural Engineering. Vol 34/3 pp 261-274.

Wiens, G.D., Lapatra, S., Palti, Y., Rexroad III, C.E., Yao, J., Welch, T.J. 2006. Gene expression and cellular immune response of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri: A model system for understanding mechanisms of teleost protective immunity. 10th International Congress, International Society of Developmental and Comparative Immunology, July 1 - 6, 2006. Meeting Abstract. Page 249.

Wiens, G.D. 2006. Bacterial kidney disease. Aquaculture Compendium. Online at Wallingford, UK: CAB International

Last Modified: 4/19/2014
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