Location: Cool and Cold Water Aquaculture Research
2006 Annual Report
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.
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.
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 (http://www.nwfsc.noaa.gov/bkd-conference), 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.
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.
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 www.cabicompendium.org/ac. Wallingford, UK: CAB International