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? From 1987 to 1989, columnaris was the most frequently reported infectious disease in the U.S. catfish industry accounting for 58% of all bacterial cases. Currently, F. columnare is considered the second most important bacterial pathogen in commercial culture of channel catfish in the U.S., second only to Edwardsiella ictaluri (NAHMS, 1997). Although columnaris negatively impacts aquaculture production around the world, bibliographical references to this species are mostly related to taxonomical status rather than virulence properties. To date, the only F. columnare sequence information available in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) corresponds to ribosomal genes. One of the main objectives within this project was to increase our genetic knowledge on F. columnare. This new genetic information would be applicable to the future development of new vaccines against columnaris. 2.List the milestones (indicators of progress) from your Project Plan. Milestone 1: Genotyping of Flavobacterium columnare isolates. Milestone 2: Developing a real time Polymerase Chain Reaction (PCR) detection protocol for F. columnare. Milestone 3: Assessing the prevalence of Flavobacterium columnare in pond raised catfish. Milestone 4: Comparing gene expression profiles between different F. columnare strains by using cDNA-AFLP analysis. Milestone 5: Sequence the whole-genome of F. columnare. 4a.What was the single most significant accomplishment this past year? A shotgun whole-genome sequencing project has been started. In order to complete this project additional funding is needed. Support is being sought from the National Science Foundation (NFS). To date, approximately 3,000 clones containing 1,2 kb of F. columnare genetic information have been sequenced. These clones yielded around 800 contings (contiguous sequences) that have been blasted in GenBank. Some putative virulence-related genes have been identified. Currently, we are responsible for more than 95% of the total F. columnare genome sequenced, however, much more additional work is needed in order to successfully complete this project. 4b.List other significant accomplishments, if any. 4.1. Optimization of total RNA extraction. We used the RNAlaterTM (Ambion) to chemically protect mRNA from degradation and to stop transcription in the cell. Following this step, two different RNA extraction protocols were evaluated: the commercial Qiagen RNeasy kit (Qiagen) and a phenol-based classical protocol. Although both methods provided high quality RNA, the phenol-based method resulted in a much higher yield. Therefore, the phenol-based protocol will be used for the rest of the study. 4.2 Optimization of total mRNA enrichment protocol. We used the MicroExpress (Ambion Inc., Austin, TX) to subtract rRNA and tRNA from the sample, therefore, enriching for mRNA. This kit appeared to be suitable for mRNA enrichment from total F. columnare RNA. However, and according to reviewer’s suggestions, specific probes against the ribosomal RNAs from F. columnare could be developed in order to enhance removal of rRNAs. In order to develop these specific probes, the 23S rDNA had to be sequenced since this information was not available in GenBank. The 23S rRNA gene from Flavobacterium columnare type strain along with the environmental isolate (ALG-530) have been fully sequenced. This new information would be used if we suspect rRNA contamination when preparing cDNA. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The major accomplishment is the generation of new sequence information from F. columnare. To date, our group is responsible for 95% of total F. columnare sequences deposited in GenBank. We expect to complete the sequencing of the entire genome in two years if additional funding is obtained. This data will help us to understand how F. columnare causes diseases and will facilitate the developing of new strategies to control columnaris infection. 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? No sequence information from the shotgun approach has been deposited into GenBank yet. Generated data will be released when at least 6X genome coverage will be achieved.