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? Edwardsiella ictaluri, the cause of enteric septicemia of channel catfish (ESC) and Flavobacterium columnare the cause of columnaris disease, are the two most important bacterial pathogens causing devastating losses to the warm water aquaculture industry in the Southern (Mississippi, Arkansas, Alabama, Louisiana and Georgia) United States. Because fish are farmed under intensive management conditions, with high stocking rates (10,000 to 12,000 fish per acre) and covering extensive pond acreages (100 to > 500 acres/farm) sudden outbreak of disease and its rapid spread could impact with disastrous economic consequences to fish farmers and the aquaculture industry as a whole. Thus, it is imperative to eliminate disease-related losses by developing efficacious vaccines, innovative, rapid disease detection techniques and surveillance strategies to combat disease. Based on these exigencies and the goals prioritized by the National Program (NP-106), our studies are aimed at: (a) elucidating the molecular genetic basis for virulence in Edwardsiella ictaluri, Flavobacterium columnare, Streptococcus iniae and S. agalactiae and analysis of molecular mechanisms of host-pathogen interactions, (b) analysis of in-vivo expression of genes encoding for virulence traits that are up and/or down regulated in the pathogen (bacteria) and host (fish)during infection, (c) determine the molecular genetic basis for antigenic variation that enables the pathogen to outwit the host's immune response, and (d) develop novel molecular methods for rapid and accurate diagnosis of enteric septicemia in catfish and columnaris disease. How serious is the problem? The three most prevalent diseases among warm water farmed food-fish, are ESC, enteric septicemia of catfish,(reported by 67% of operations); columnaris disease (reported by 50% of operations); and winter kill (reported by 33% of operations), (catfish 2003, released by the USDA). Infectious disease continues to be the preeminent cause for production losses in the US aquaculture industry. Why does it matter? The US aquaculture industry is recognized as the fastest growing agricultural enterprise, netting over a billion dollars in profits (report of 2000 aquaculture census: http://www.nass.usda.gov/mt/pressrls/misc/aquacult.text). Growth in the domestic aquaculture industry has been propelled by a parallel consumer demand for fish as a healthy nutritional source of protein. Thus, US per capita consumption of fish has rapidly risen. Because of its cutting edge technology and cadre of specialized aquaculture scientists, USA is well positioned to keep the domestic aquaculture industry robust, while reducing foreign fish imports. Reducing or eliminating losses due to disease is vital from an economic perspective and for sustaining the growth of the industry. In its broad mandate, the USDA aquaculture national program 106 has targeted microbial genomics as a high priority area of research. To address this need, our project is geared to elucidate heretofore unknown intricate virulence mechanisms of E. ictaluri, F. columnare S. iniae and S. agalactiae using novel molecular biological methods. 2.List by year the currently approved milestones (indicators of research progress) This research project (# 6420-32000-020-00D), one of four submitted by the Aquatic Animal Health Research Unit (AAHRU), is focused on molecular analysis of virulence determinants of select bacteria in fish diseases. Within the context of this broad title are included four milestones to be addressed within the projected tenure of the prohect, beginning in November 2004. Milestone 1. 2004-2008: Identify, clone and characterize adhesins of Edwardsiella ictaluri. Milestone 2. 2004-2008: Characterize novel genes in (host) channel catfish and concurrently, those genes expressed (up/and/or down regulated) in (pathogen) Edwardsiella ictaluri. Milestone 3. 2004-2008: Characterize genes expressed by Flavobacterium columnare under in-vivo compared to in-vitro culture conditions. Milestone 4. 2004-2008: Use genomic and pathogenetic variation to identify putative virulence genes of Streptococcus iniae and Streptococcus agalactiae. 4a.List the single most significant research accomplishment during FY 2006. Enteric septicemia of catfish, caused by E. ictaluri, columnaris disease, caused by F. columnare and motile aeromonad septicemia, caused by Aeromonas hydrophila are three most prevalent diseases caused by Gram negative bacteria with profound economic impact as before mentioned. Since all three bacteria are ubiquitous in the aquatic environment and disease symptoms are not pathognomonic, disease could be confounded by simultaneous co-infection with any one or more bacteria. Traditional diagnosis using culture techniques require several (5 to. 8)days to arrive at a definitive diagnosis. A novel multiplex-polymerase chain reaction (m-PCR) technique developed for the first time in our laboratory will enable simultaneous and accurate identification of the three organisms with a minimum of cost, effort, and time (within hours of observing signs of disease or mortality in a farm). This test will impact by enabling aquaculture farmers to make prompt management decisions to avert the spread of disease from pond to pond or from farm to farm and to quickly salvage vulnerable uninfected stock by vaccination or through surveillance measures. 4b.List other significant research accomplishment(s), if any. In other project related research, notable accomplishments were made on efforts to address catfish gene (up or down) regulation subsequent to E. ictaluri infection (Milestone 2). Through a novel suppression-subtractive hybridization technique, in excess of 200 up-regulated catfish genes were identified following E. ictaluri infection for the first time. Further validation of select genes by cloning, sequencing and expression have enabled successful characterization of genes encoding for peroxiredoxin 6, matrix metalloproteinase-9 and the CD59 receptor (i.e., membrane inhibitor of complement-mediated reactive lysis). These gene sequences have been deposited in the NCBI data bank and made available for use by the scientific community, while additional catfish genes are being analyzed. Three proteins of E. ictaluri that are expressed under in-vivo conditions have also been identified and presently subjected to amino acid sequence analysis. Gene expression in F. columnare was evaluated for the first time (Milestone 3). Eight putative virulence genes of F. columnare have been successfully identified. Amplified fragment length polymorphism (AFLP) on S. iniae was completed (Milestone 4). 4c.List significant activities that support special target populations. None. 4d.Progress report. Not applicable. 5.Describe the major accomplishments to date and their predicted or actual impact. Three test systems based on novel molecular biological approaches were developed, sensitivity/specificity tested under simulated field conditions, validated and made available for rapid and accurate detection of the three most important bacterial pathogens (E. ictaluri, F. columnare and A. hydrophila) that have a profound economic impact on the warm water aquaculture industry. These tests are presently being used in our (AAHRU) laboratory and complete technology made available via publications and presentations to the scientific community and to interested aquaculture commodity groups. Putative virulence determinants of E. ictaluri and F. columnare were partially characterized and additional virulence determinants and/or related moieties encoded by yet unidentified genes will be elucidated over the tenure of the project as detailed in this project. Several important genes that are up and or down regulated in the catfish (host) during infection with E. ictaluri, as well as genes expressed in E. ictaluri (the pathogen) during infection have been identified for the first time, as have several virulence genes of F. columnare. The occurrence of the phenomenon of antigenic variation in S. iniae and S. agalactiae (emerging panzootic pathogens) will be tested and molecular mechanisms involved will be evaluated. Predicted or actual impact: Fish farmers operate on narrow profit margins and even a modest drop in production could have a serious impact on the sustainability of the burgeoning U.S. warm water aquaculture industry. Thus, when confronted with a disease outbreak, prompt and accurate diagnosis of disease is a prerequisite for timely intervention to control and/or eradicate the disease. The test systems developed in our research will address this problem and help to substantially cut-down disease-related losses. To date several functional as well as house-keeping genes of E. ictaluri and F. columnare have been identified and the sequence information made available in the (NCBI) public domain. This information will expand the knowledge-base on virulence genes of important fish pathogens; help accurately annotate the functional basis of gene expression in ongoing whole genome projects and related research. Discovery of putative gene and gene products will spawn the development of rapid specific tests for disease diagnosis; enable identification of potential vaccine candidate-antigens and development of receptor analogs as therapeutics. Our research will benefit other aquaculture researchers, fish farmers, aquaculture commodity-groups and industry personnel. Overall, our research will benefit the consumer (end-users) that demand for a wholesome, value-added nutritional product that is available in fish and fish products (Omega-3 fatty acids) and help bolster the US aquaculture industry. 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? Three molecular biology based sensitive/specific test systems (PCR based, multiplex-PCR and real time PCR) were developed and while being used for routine disease diagnosis in our laboratory; the technology has been disseminated to other aquaculture disease diagnostic laboratories and industry through scientific publications, and presentations at scientific forums/workshops as indicated below. Several new monoclonal antibody reagents were developed against E. ictaluri and F. columnare antigens. While some monoclonal antibody- producing cell lines have been deposited in the American Type Culture Collection, additional cell lines are being evaluated as specific antibody reagents for use in diagnostic test systems such as the indirect fluorescent antibody test (IFAT), the enzyme-linked immunoabsorbent assay (ELISA) and other aquaculture research since such reagents are meager or unavailable for use by researchers. As indicated above, several new gene sequences have been deposited in the data bank (NCBI) and made available to scientists throughout the world. When is the science or technology likely to become available to the end-user (industry, farmers, other scientists)? Results that are generated throughout all phases of our research have been published in scientific journals and presented at scientific forums. Gene sequence information will be accessible through the public domain (NCBI) by all interested scientist and monoclonal antibody-producing cell lines and other tissue culture cell lines will be deposited in the ATCC repository for use by any scientist. The immediate benefits of our research will be realized by other scientists. Research in this Cproject is linked to the research objectives of inhouse project 6420-32000-019-00D, entitled "Vaccinology and Immunity in Aquatic Animals", with scientists from this unit; Auburn University, Department of Fisheries and Allied Aquacultures who are working on pathogenicity and the development of vaccines for immunization against fish diseases; and USDA scientists at Chestertown, MD. Regional: Scientists at Oxford, MS; Scientists working on fish diseases at the Mississippi State University, Louisiana State University, and University of Arkansas. Our studies will complement the molecular biological studies (whole genome projects on E. ictaluri and F. columnare) presently being conducted at the Mississippi State University. National: ARS scientists at Orono, and Franklin, ME; Wyndmoor, PA; Dover, DE; ARS researchers at Leetown and Shepherdstown, WV. Also Internationally: scientists working on fish diseases in China, Japan, Thailand, Taiwan, India and the Philippines. What are the constraints if known, to the adoption and durability of technology products? The only constraint is the cost involved in purchasing reagents, expensive equipment and the need for trained laboratory personnel to conduct the diagnostic tests. Hence, molecular-based tests could only be conducted in a laboratory. However, well equipped laboratories capable of conducting these tests are available in USDA regional laboratories and laboratories in universities where fisheries and allied aquaculture programs are purveyed. Review Publications Yeh, H., Shoemaker, C.A., Klesius, P.H. 2005. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri. Journal of Microbiological Methods 63(1): 36-44. Panangala, V.S., Shelby, R.A., Shoemaker, C.A., Klesius, P.H. 2005. Rapid tests for detection of two major fish pathogens Edwardsiella ictaluri and Flavobacterium columnare. 6th Symposium on Diseases in Asian Aquaculture Proceedings. Colombo Plaza Hotel, Colombo, Sri Lanka. October 25-28, 2005. Panangala, V.S., Klesius, P.H., Evans, J.J., Shoemaker, C.A. 2006. Beneficial use of attenuated vaccines in aquaculture. 31st Annual Eastern Fish Health Workshop. March 28-30, 2006. Charleston, SC. page 23. Yeh, H., Klesius, P.H. 2006. Diferential channel catfish ovary cell gene expression after experimental infection with Edwardsiella ictaluri. 106th Annual Meeting of the American Society for Microbiology Orlando, FL. Abstract No. Z-005. Panangala, V.S., Shelby, R.A., Shoemaker, C.A., Klesius, P.H., Mitra, A., Morrison, E.E. 2006. Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare. Diseases of Aquatic Organisms, 68: 197-207. Panangala, V.S., Shoemaker, C.A., Mcnulty, S.T., Arias, C.R., Klesius, P.H., 2006. Intra- and Interspecific phenotypic characteristics of fish-pathogenic Edwardsiella ictaluri and Edwardsiella tarda. Aquaculture Research, 37: 49-60.