2007 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research project is to identify virulence factors in the genome of Flavobacterium psychrophilum by use of DNA sequencing. Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease. The disease causes significant production losses of rainbow trout and novel methods are urgently needed for control of this disease. Knowledge of the F. psychrophilum genome should serve as a foundation for future development of new vaccines, improved diagnostic tests, and novel therapeutics. 1b.Approach (from AD-416) Genomic DNA will be isolated from a virulent clone of Flavobacterium psychrophilum strain CSF-259-93 and random, large-insert and small-insert DNA libraries will be created. The DNA libraries will be used to generate clones for 10,000 sequencing reactions for low-coverage sequence analysis of the genome. Sequence data will be organized into contigs and preliminary annotation will be performed using the ERGO bioinformatics system developed by Integrated Genomics. Preliminary annotation will allow assessment of the metabolic and genetic organization of Flavobacterium psychrophilum. The partially assembled genome sequence will be available on the NCCCWA web site and a reference clone bank established. 3.Progress Report 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 vaccine development have been ineffective and antimicrobial resistance is of concern. This year we reported the completed genome of Flavobacterium psychrophilum. The genome consists of a single circular chromosome of 2,900,735 bp with a G+C content of 32.49%. A set of 19 highly similar genes were identified that contain variable numbers of a short repeat. These repeats have homology to leucine-rich motifs. A PCR assay was developed to distinguish between genes containing various numbers of repeats and this assay was used to show that this locus is highly polymorphic between strains. One of these genes, designated F. psychrophilum repeat protein N (frpN), was cloned and recombinant protein purified. This protein was weakly immunogenic in rainbow trout and did not induce a protective immune response against challenge. The complete genome sequence is the first step toward a better understanding of metabolic and pathogenic processes, and the identification of potential vaccine candidates. The identified genes are the basis of ongoing vaccine studies. The completion of the genome sequence was the stimulus for a workshop titled Flavobacterium 2007 held in Shepherdstown WV on May 2-4th that was attended by over 70 scientists from eight countries. This work also addresses research outlined in the National Program 106 Action Plan. Integrated Aquatic Animal Health. Goal 1. Microbial Genomics. The ADODR is in frequent contact with the cooperator through phone calls, email, and annual meetings in addition to receipt of written reports.