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? Edwardsiella ictaluri, the cause of enteric septicemia of channel catfish is the leading cause of mortality in catfish in the USA and worldwide. Flavobacterium columnare the cause of columnaris disease, is second only to Edwardsiella ictaluri in its repute as a devastating pathogen causing widespread infection in intensive fish-farming states (Mississippi, Arkansas, Alabama, Louisiana and Georgia). Fish are farmed under intensive management conditions, with high stocking rates (10,000 to 12,000 fish per acre) and covering extensive pond acreages. Thus, sudden outbreak of disease and its unchecked spread could impact with profound economic consequences to fish farmers and industry as a whole. It is compelling and important to eliminate disease-related losses by developing efficacious vaccines, innovative, rapid disease-detection techniques and efficient strategies to prevent the spread of disease. Within the context of the National Program (NP)106 priorities, the focal points of our studies are: (a) elucidate the molecular genetic basis for virulence in Edwardsiella ictaluri and Flavobacterium columnare and analyze mechanisms of host-pathogen interactions; (b) analyze the in-vivo expression of genes encoding for virulence traits that are up and/or down regulated during infection, both in pathogen (bacteria) and host (fish); (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 (ESC) and columnaris disease. How serious is the problem? The three most prevalent fish diseases among warm-water farmed food-fish, are enteric septicemia of catfish (ESC) (66.6% of operations); columnaris disease (50.4% of operations) and winter kill (32.9% of operations) (Catfish 2003, released by the USDA). Infectious disease takes precedence over all other contributing causes for production losses in the aquaculture industry. Why does it matter? According to the 2000 aquaculture census (http://www.nass.usda.gov/mt/pressrls/misc/aquacult.txt), the US aquaculture is nearly a billion dollar industry and is slated as one of agriculture’s fastest growing sectors. The 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 aquaculture industry robust to meet the growing public demand for fish as a healthy nutritional source. Thus, eliminating the losses due to infection is vital and compelling both from a nutritional and economic perspective. In its broad mandate, the USDA aquaculture national program 106 has highlighted microbial genomics as a high-priority area of research and has emphasized the “critical need to obtain a better understanding of the molecular basis by which microbial pathogens cause disease in and interact with their host”. A thorough review of the literature revealed a void in the scope of research we have detailed in our research project. Thus the sparse, incomplete or lack of information at the molecular genetic level on fish-pathogens underscores the need for this research. 2.List the milestones (indicators of progress) from your Project Plan. This research project, 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 research project, beginning in November, 2004. These milestones include: (1) Identify, clone and characterize the putative adhesins of the ESC causing pathogen, Edwardsiella ictaluri; (2) Characterize novel genes in the host, channel catfish and concurrently in vivo expressed genes of the pathogen, Edwardsiella ictaluri during infection; (3) Characterize genes expressed by Flavobacterium columnare (the causative agent of columnaris disease) under in vivo compared to in vitro culture conditions; and (4) Use molecular techniques to elucidate genomic and pathogenetic variations and identify putative virulence genes of Streptococcus agalactiae and Streptococcus iniae. 4a.What was the single most significant accomplishment this past year? Development of a rapid, sensitive and cost-effective immunofluorecent test for simultaneous diagnosis of Edwardsiella ictaluri and Flavobacterium columnare infection in fish was a significant accomplishment. This test was validated using 303 samples (derived from kidney, brain and nares) from both experimentally and naturally infected fish and found to be sensitive and specific for detection of Edwardsiella ictaluri and Flavobacterium columnare compared to the bacteriological culture (gold standard) which requires a minimum of 3-4 days for definitive disease diagnosis. The rapid diagnosis of disease is vital in the context of making important management decisions promptly since fish are farmed under intensive management conditions with high stocking rates and covering extensive pond acreages. Under such conditions, the sudden outbreak of disease and its unchecked spread could be economically devastating. The dual immunofluorescence test is an efficient, versatile tool for the rapid diagnosis of ESC and columnaris disease simultaneously with a minimum of cost. 4b.List other significant accomplishments, if any. Research that was in progress prior to implementation of the current research project proceeded in parallel with the above mentioned studies that were realistically initiated following final approval of the research project in November 2004. Thus, other significant accomplishments on ongoing experiments included: (1) Four independent experiments conducted to assess the efficacy of six field isolates of Edwardsiella ictaluri to mediate cross protection in controlled challenge studies revealed differences among isolates in the degree of immunity conferred. This study was completed mid year, and a manuscript is in preparation. (2) To determine whether Edwardsiella originating from disparate environments show adaptive pleiotropic alterations that are reflected as appreciable phenotypic differences, phenotypic and genetic characteristics of 18 Edwardsiella ictaluri and 9 Edwardsiella tarda isolates from outbreaks of fish disease in several geographic locations were examined on the basis of several well established phenotypic and genotypic parameters. These studies revealed definitively that Edwardsiella ictaluri displays a clonal bacterial population structure wherein essential phenotypic traits are maintained, possibly for a selective survival advantage, whereas the closely related Edwardsiella tarda is genetically and phenotypically more polymorphic. Additionally, restriction enzyme cleavage sites, predicted recognition sequences, fragment size and positions of polymorphism within the 16S-23S intergenic spacer regions were delineated for both Edwardsiella ictaluri and Edwardsiella tarda. New sequence information representing twenty seven new sequences (of the intergenic spacer regions of Edwardsiella ictaluri and Edwardsiella tarda) each identified by a GenBank accession number was added to the data base. (3) A total of 15 new Flavobacterium columnare were fingerprinted and added to the existing AAHRU database thus, bringing the total number of Flavobacterium typed by AFLP, sequenced and deposited to forty-five. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Our research has enabled the plasmid mediated transformation of Edwardsiella ictaluri to identify the host-pathogen interactions which are prerequisite for pathogenesis and disease. The molecular mechanisms of pathogen adherence to the host tissues will be elucidated. In concert, the genes encoding for these adhesins will be cloned and sequenced. The genes will be mutated by site-directed mutagenesis and the adhesion deficient mutants will be compared with the parent in, in vitro experiments with tissue culture cells derived from catfish and in, in vivo experiments with live catfish. Differentially expressed cDNA libraries of Edwardsiella ictaluri have been successfully constructed using suppression subtractive hybridization methods. The subtracted target cDNA has been specifically amplified by nested PCR and amplified products ligated into plasmid vectors and chemically transformed into Escherichia coli. Transformants were randomly selected and the mapped DNA extracted from isolated colonies were submitted for sequencing at the USDA, Mid South Genomics Laboratory for sequencing. Significant strides have been made in the quest for channel catfish gene regulation. Through suppression-subtractive hybridization, differentially expressed channel catfish cDNA libraries were constructed and more than 200 genes up-regulated following Edwardsiella ictaluri infection have been identified. In other studies, significant accomplishments were made in attempts to sequence the complete 23S rDNA gene of Flavobacterium columnare. This information will be available in the public domain. A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region of the ribosomal operon was developed. The method was evaluated for specificity and sensitivity and found to be able to detect as low as seven colony forming units and is specific in that, it detects only the target DNA of Flavobacterium columnare. The test was proven to be efficient in field studies. The immediate impact of our research is to expand the knowledge-base on hitherto unknown functional genes encoding for virulence traits of major bacterial fish pathogens that cause devastating losses in the aquaculture industry in the USA and world wide. Discovery of putative gene and gene products will spawn the development of rapid specific tests for disease diagnosis, efficient genetics-based vaccines and potential competitive inhibition of pathogens via intervention by receptor analogs. Our research will benefit other scientists, fish farmers, aquaculture commodity-groups and industry personnel. Overall, our research will benefit the consumers (end-users) that demand for a wholesome, value-added nutritional product that is available in fish and fish products. 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? Overall, our research within this short span of eight months has generated a wealth of sequence information on several structural and house-keeping genes of Edwardsiella ictaluri, Flavobacterium columnare, Streptococcus iniae and Streptococcus agalactiae, which have been deposited in the GenBank as indicated above. Several new molecular-based and conventional diagnostic test procedures were developed and publications describing these techniques have been made available to the scientific community via refereed scientific journals, and at scientific forums. Several monoclonal antibodies (reagents) developed by AAHRU researchers against Edwardsiella ictaluri have already been deposited in the American Type Culture Collection repository and new monoclonal antibodies presently developed against Flavobacterium columnare, will shortly be available for deposition as specific monoclonal antibody generating cell-lines. It may be pertinent to note that these will be the first such reagents to be made available, since there is a lack of secondary antibody reagents required for use in diagnostic test systems such as the enzyme-linked immunoabsorbent assay (ELISA) and for use in other aquaculture research. The immediate benefits of our research will be realized by other scientists at the USDA/ARS-AAHRU, Auburn, AL. Research in this research project is linked to the research objectives of research project 6420-32000-019-00D entitled, Vaccinology and Immunity in Aquatic Animals, by this unit; Auburn University Department of Fisheries and Allied Aquacultures who are working on pathogenicity studies 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 presently being conducted at these institutions. National: ARS scientists at Orono and Franklin, ME; Wyndmoor, PA; Dover, DE; ARS researchers at Leetown and Shepherdstown, WV; and Internationally: scientists who are working on fish diseases in China, Japan, Thailand, Taiwan, India and the Philippines. At present we do not envisage any constraints to the adoption and/or durability of the technology products. Review Publications Panangala, V.S., Shoemaker, C.A., Klesius, P.H. 2005. Phenotypic and genotypic comparisons of edwardsiella ictaluri and edwardsiella tarda isolates from fish. Aquaculture America Conference. Panangala, V.S., Shoemaker, C.A., Vansanten, V., Mcnulty, S.T., Arias, C. 2004. Polyphasic characterization of edwardsiella ictaluri and e. tarda isolates from fish. American Society for Microbiology Branch Meeting. Panangala, V.S., Vansanten, V.L., Shoemaker, C.A., Mcnulty, S.T., Arias, C.R., Klesius, P.H. 2005. Intra-and interspecific phenotypic and genotypic comparison of fish-pathogenic edwardsiella ictaluri and edwardsiella tarda. American Society of Microbiologists Abstracts.