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? Channel catfish aquaculture represents the largest venture of its kind in the U.S.; however, its viability is threatened by increasing disease losses. Commercial catfish production takes place in 13 states, but is concentrated in the Mid-South Area, particularly the Delta region of northwest Mississippi. In 2003 the industry produced 300 million kilograms of foodsize fish, valued at approximately $400 million. However, the overall economic impact of U.S. catfish production vastly exceeds this figure. In Mississippi alone, the industry encompasses roughly 400 farms, 45,000 hectares of water and employs 7,000 workers. Mississippi accounts for approximately 60% of total foodsize fish production and is also the largest producer of fingerling “seed-stock.” When ancillary service industries are considered, the Mississippi industry contributes approximately $2 billion to the well being of this economically depressed region. Despite increased production, profitability of the catfish industry is declining. Since its inception more than 40 years ago, disease-related losses have represented a major impediment to economic performance. Losses from recognized infectious and environmental diseases have escalated as culture practices have become more intensive and there has also been an emergence of several previously unknown disease entities. It is estimated that disease-related mortalities account for approximately 45-50% of all losses incurred on farms and may account for as much as $100 million annually in direct economic impact. In the past 3 years, decreased production resulting from disease, in conjunction with historically low fish prices, competition with foreign imports, and rising feed costs has forced approximately 20% of farm acreage out of production. Without substantial improvements in fish health management and production efficiency, the future of commercial catfish farming will remain in jeopardy. This project addresses priority issues for increasing fish health, production efficiency and profitability of commercial catfish farming. The overall objective is to address current and emerging fish health problems by developing improved disease diagnostic methods and comprehensive management strategies. We have addressed these goals using a multidisciplinary approach that focuses on practical, production-oriented methods that integrate existing strengths of the National Warmwater Aquaculture Center with state-of-the-art technologies. This research directly addresses several components of the National Program 106 Aquaculture Action Plan, such as: Component 2 (Integrated Aquatic Animal Health Management) Problem a (Pathogen Identification and Disease Diagnosis), Problem b (Vaccines and Medicines), Problem c (Immunology and Disease Resistance), Problem e (Epidemiology), Problem g (Aquatic Animal Health Medicine), Problem h (Immune System Enhancement – Nutrients and Immunostimulants), as well as Component 4 (Growth and Development, and Nutrition). Taken as a whole, this project aims to develop an integrated system of managing catfish health problems and therefore all of the objectives ultimately address the primary stated goal of Problem g (Aquatic animal health medicine), which is to “improve health management practices currently used in aquaculture.” All objectives are focused on the area of fish health management and diagnostic medicine to establish new effective measures for the detection, prevention, and treatment of disease. Reducing disease-related costs will increase net profitability and help insure economic viability of the catfish industry. Advances in the understanding of disease transmission, pathogenesis, and epidemiology, along with the development of state-of-the-art diagnostic technologies, will provide benefits to the scientific community and fish health providers throughout the U.S. The program is closely integrated with the USDA-ARS Catfish Genetics Research Unit and will enhance current projects involved in the genetic selection of disease-resistant lines of channel catfish. Expected products will ultimately be the development of best management practices (BMPs) that increase production efficiency, promote fish health, and reduce economic risks associated with disease losses. Ultimately, American consumers will be the major beneficiaries through the production of an inexpensive, safe, domestically produced seafood product that will help offset the large U.S. trade deficit in seafood commodities. 2.List by year the currently approved milestones (indicators of research progress) Objective 1: Develop diagnostic tools to detect and monitor diseases in commercially raised channel catfish and determine virulence factors associated with those diseases. Hypothesis 1: A real-time PCR assay can be developed to study the epidemiology of proliferative gill disease. Milestones FY 2005 – Validate the specificity of a pair of H. ictaluri PCR primers and initiate the development of methodologies for isolating spores from pond water (filtration or imuno-capture). FY 2006 – Develop real-time PCR assay and optimize conditions for its use. FY 2007 and 2008 – Conduct field investigations using the PCR assay and optimize conditions for its use. FY 2009 – Objective complete. Hypothesis 2: A real-time immuno-PCR assay can be developed to study the epidemiology of enteric septicemia of catfish. Milestones FY 2005 – Optimize real-time PCR assay for E. ictaluri and develop immuno-capture methodologies. FY 2006 – Conduct laboratory trials and optimize conditions for use of assay. FY 2007 and 2008 – Conduct field trials and optimize conditions for use of assay. FY 2009 – Objective complete. Hypothesis 3: Early stages in the pathogenesis of enteric septicemia can be elucidated by examining differential protein expression by Edwardsiella ictaluri exposed to channel catfish intestinal mucus. Milestones FY 2005 – Evaluate growth of Edwardsiella ictaluri in water and catfish intestinal mucus. FY 2006 – Characterize protein expression under above growth conditions using electrophoresis. FY 2007 – Develop antibodies to proteins of interest identified above. FY 2008 – Evaluate protection derived from isolated proteins and conduct challenges. FY 2009 – Objective complete. Hypothesis 4: Strain differences in Edwardsiella ictaluri and Flavobacterium columnare can be identified by genetic variation among isolates. Milestones FY 2005 – N/A FY 2006 – Optimize ERIC, Rep, and BOX PCR methodologies for Edwardsiella ictaluri and Flavobacterium columnare. FY 2007 and 2008 – Analyze archived strains of above bacteria for genetic variation. FY 2009 – Conduct challenges. Hypothesis 5: Edwardsiella ictaluri strains isolated during epizootics at abnormally high water temperatures vary in growth and genetic features from those isolated at normal temperatures. Milestones FY 2005 – N/A FY 2006 – Optimize culture methods for growth of archived E. ictaluri strains growing at higher than expected temperatures. FY 2007 – Determine growth characteristics of above bacterial isolates and perform challenge trials on channel catfish fingerlings. FY 2008 – Objective complete. Objective 2: Develop fish health management procedures to control economically important diseases of channel catfish. Hypothesis 1: Acute and chronic Bolbophorus sp. trematode infections decrease growth, production efficiency, and health status of channel catfish fingerlings. Milestones FY 2005 – Evaluate effects of acute Bolbophorus sp. infections on channel catfish. FY 2006 and 2007 – Evaluate relationship of acute Bolbophorus sp. infection on susceptibility to enteric septicemia in pond studies. FY 2008 and 2009 – Evaluate the effects of chronic Bolbophorus sp. infection. Hypothesis 2: Method of vaccine preparation and bacterial exposure influence the efficacy of AQUAVAC-ESC® in challenge trials. Milestones FY 2005 – Optimize conditions for AQUAVAC-ESC® vaccination and conduct challenge trials. FY 2006 – Objective complete. Hypothesis 3: Feed restriction and inclusion of natural feeds to the diet enhance resistance to enteric septicemia of catfish and improves vaccine efficacy. Milestones FY 2005 – Establish validity of restricted feeding practices for control of enteric septicemia. FY 2006 and 2007 – Evaluate interaction of restricted feeding and AQUAVAC-ESC® vaccination on survival of channel catfish exposed to E. ictaluri infection. FY 2008 and 2009 – Conduct field trials. Hypothesis 4: AQUAVAC-ESC® and AQUAVAC-FC® used in combination will reduce losses of channel catfish fingerlings to enteric septicemia and columnaris disease. Milestones FY 2005 - Optimize conditions for AQUAVAC-FC® vaccination and conduct challenge trials. FY 2006 – Evaluate efficacy of combined AQUAVAC-ESC® and AQUAVAC-FC® vaccination on survival of channel catfish. FY 2007 – Continue to evaluate efficacy of combined AQUAVAC-ESC® and AQUAVAC-FC® vaccination on survival of channel catfish. FY 2008 and 2009 – Conduct field trials. Objective 3: Determine factors associated with emerging diseases in pond-raised channel catfish. Hypothesis 1: An unidentified toxin is responsible for visceral toxicosis of catfish (VTC). Milestones FY 2005 – Isolate and characterize toxin responsible for development of VTC. FY 2006 – Examine immune system recognition of VTC toxin and develop antibodies against the toxin. FY 2007 – Develop ELISA method for detection of VTC toxin. FY 2008 and 2009 – Conduct field trials. Hypothesis 2: Iron deficiency is responsible for development of channel catfish anemia (CCA). Milestones FY 2005 – Examine response of channel catfish suffering from natural development of CCA to parenteral iron and determine the lifespan of the catfish erythrocyte. FY 2006 – Evaluate effect of feeding an iron deficient diet on development of CCA-like disease. FY 2007 and 2008 - Examine possible mechanisms for the disturbance of normal iron metabolism in the pathogenesis of CCA. FY 2009 – Objective complete. Hypothesis 3: A new streptococcal disease causes losses of brood-sized channel catfish. Milestones FY 2005 – Characterize the streptococcal bacteria isolated from an emerging disease of channel catfish and fulfill Koch’s postulates. FY 2006 – Objective complete. Objective 4: Use epidemiological methods to investigate new and emerging diseases, and to identify environmental and management factors that influence the onset and severity of disease outbreaks. Hypothesis 1: A systematic data-collection system can be used to identify risk factors associated with the diseases visceral toxicosis of catfish and channel catfish anemia. Milestones FY 2005 – Develop infrastructure for data collection, including hiring of essential personnel and construction of database. FY 2006 – 2008 – Collect field data. FY 2009 – Evaluate associations from collected data. 4a.List the single most significant research accomplishment during FY 2006. The trematode, Bolbophorus spp., has been associated with high mortalities and production loss in commercial catfish. Research was conducted to establish the prevalence, severity and economic impact of this disease on a commercial catfish operation. Of the 38 ponds sampled, 17 were categorized as negative, 6 as light, 6 as moderate and 11 as severe. Fish in the negative category consumed 73.4 lbs/ac/day, and fish categorized as light, moderate and severe consumed 62.2, 47.5 and 47.2 lbs/ac/day, respectively. Compared to trematode negative ponds, ponds in the light, moderate and severe categories produced 16.8%, 36.4% and 44.5% less fish weight per acre, respectively. Net returns from ponds in the light category were reduced by 80.8%. Ponds in the moderate and severe category produced net losses of $506 and $631 respectively. This data showed trematode infections decreased production, even with light infections. Tests were conducted in commercial ponds to determine the efficacy of copper application to ponds to eradicate snails. Results in ponds demonstrated that copper sulfate pentahydrate (2.5 and 5.0 mg/L CSP) was effective in killing snails around the margins of the pond and throughout the water column, but had a negative impact on fish health. These trials indicate copper application to pond water can be used with caution as an effective treatment against snails. 4b.List other significant research accomplishment(s), if any. On an annual basis, the Aquatic Diagnostic Laboratory processes between 1,600 and 2,000 case submissions. Routine services performed include gross necropsy, histopathology, bacteriology, virology, mycology, parasitology and water quality evaluation. The aquatic diagnostic laboratory works closely with producers to provide and evaluate treatment recommendations, monitor disease trends, provide surveillance for and investigation into the causes of new and emerging disease, and provide field service investigations. The objectives of the fish health research program are focused on the development of management programs designed to improve production efficiency and establish new effective measures for the detection, prevention and treatment of disease. Significant research accomplishments were associated with the control and pathobiology of bacterial and parasitic diseases, improved diagnostic technologies and diseases of unknown etiology. Visceral toxicosis of catfish (VTC) is a disease of unknown etiology and is seen primarily in market size and brooder fish. Since spring 1998, outbreaks have been recognized as seasonal and occur in the early spring and late fall when pond temperatures are between 18-22 °C. In the spring of 2005, assays were performed using sera from affected fish that strongly suggested botulism type E as the cause of VTC. Because of the limited supply and lability of affected serum, further assays for botulism could not be performed at this time. With a new outbreak in the spring of 2006, more bioassays with catfish were performed which defined botulism as the cause of VTC. The validity of this assay was confirmed by The Centers for Disease Control and Prevention in Atlanta, GA., by Mass Spectrometry. Channel catfish anemia (CCA) is another disease of unknown etiology and generally affects market size fish. CCA was show to be caused by iron deficiency. While dietary iron supplements did not elevate hematocrit values, parenteral administration of iron dextran was shown to promote recovery. The data suggests iron deficient anemia associated with CCA is related to interference in the absorbtion or transport of iron and not a dietary deficiency. In unrelated laboratory trials, symptoms consistent with CCA developed in channel catfish fingerlings fed a commercial channel catfish diet. Summary of hematocrit values showed that approximately 14% of fish sampled had hematocrit values of 10% or less. None of the fish fed a standard reference diet developed symptoms consistent with anemia. Supplementing the diet with lysine, ferrous sulfate, folic acid or vitamin C was not shown to correct the development of this condition. This conditions appears to mimic the characteristics of CCA and is one potential cause of this disease. In October 2005, the USFDA approved the use of Aquaflor® (Schering-Plough Animal Health) medicated feed for the control of ESC in catfish. In preceding years, MSU-CVM conducted the efficacy, palatability, and safety trials in support of this approval. Field usage of this antibiotic began in April 2006 in the Mississippi Delta. Farmers are reporting that Aquaflor® treated fish are feeding during ESC outbreaks, and there is decreased mortality in these fish. Decreased mortality has also been reported in ponds that are experiencing concurrent outbreaks with ESC and columnaris. The potential impact is that Aquaflor® will help increase catfish productivity during ESC season. Restricted feed practices and vaccination where also shown to significantly reduce ESC related mortalities by 50-60%. The combined use of these management options can greatly reduce ESC losses on commercial operations and improve production efficiency. Disease monitoring and risk assessment programs focused on enteric septicemia of catfish, columnaris disease, trematode infections, and proliferative gill disease. Immuno-capture beads were shown to be effective in recovering E. ictaluri and F. columnaris pathogens from raw pond water. Quantitative real time PCR was not shown be more sensitive than standard plate count procedures in enumerating captured bacteria. A quantitative real time PCR for the detection of Hennguya ictaluri spores, the causative agent of PGD, has been developed with a level of sensitivity of 10 spores per ml of pond water. Current studies are utilizing H. ictaluri PCR primers and probes to characterize the pathobiology of the parasite in catfish. These technologies and monitoring programs provide a means of monitoring pathogen levels in production systems and may provide a method of predicting potential disease epizootics. To assist disease monitoring programs, a computerized database has been constructed to collect information on feed consumption, stocking rates, harvest rates, mortality rates, water quality, and disease diagnoses. This project is being conducted in collaboration with a large commercial catfish producer and represents a major step forward in cooperation with the catfish industry. Four years of data will be collected and the information gained will be used to examine interactions between disease incidence, production parameters, and the environment. Virulence factors for E. ictaluri and F. columnare are being determined by 1D and 2D gel electrophoresis by extracting membrane proteins from bacteria grown in media, pond water and in vivo. Protocols, utilizing immuno-capture beads, have been developed to recover of bacterial pathogens from host tissues while maintains in vivo expression of proteins. Isolates of F. columnare and E. ictaluri from diagnostic case submissions have been collected, confirmed and archived. These isolates represent the majority of geographic regions in the southern United States that commercially produce channel catfish. ERIC/rep/BOX PCR conditions have been optimized to identify genetic variations in these isolates. Pulse field gel electrophoresis has identified three subgroups of F. columnare and disease challenge trials are being conducted to determine the virulence of each sub-group. 4c.List significant activities that support special target populations. It is estimated that disease-related mortalities account for 45-50% of all losses incurred on farms and may account for as much as $100 million annually in direct economic impact. Production inefficiencies related to management and disease losses disproportionately affect small farms because they are more susceptible to economic impacts related to interruption of cash flows. The USDA Census of Aquaculture conducted in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the 1,370 catfish farms in the United States, 38% (515) reported annual revenues of less than $25,000. The profitable operation of small farms is dependent on improvements in disease management to curtail monetary losses associated with disease. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. The catfish health research project was initiated on 08/01/02 to develop a comprehensive disease management program that addresses disease related problems that threaten the economic wellbeing of commercial channel catfish farming. The current project plan, approved in 2004, continues to enhance existing disease research and diagnostic programs at the Thad Cochran National Warmwater Aquaculture Center. Major accomplishments to date involve investigation of the prevalence, severity, and economic impact of Bolbophorus sp. trematode infections, as well as the development of practical management strategies. Other accomplishments include the determination of a link between iron deficiency and channel catfish anemia, identification of a new streptococcal disease of catfish, identification of the toxin responsible for visceral toxicosis of catfish, and the development management practices utilizing medicated feeds, restricted feeding practices and vaccines to control bacterial infections. Accomplishments expected over the life of the project are to:. 1)develop state-of-the-art diagnostic tools and procedures to detect and monitor diseases in commercially raised channel catfish;. 2)develop best management practices for improving fish health and increasing the profitability of catfish farming by limiting the economic impact of infectious and non-infectious diseases;. 3)develop field and laboratory disease challenge models used to study pathogenic mechanisms of disease and develop effective control strategies;. 4)maintain a field service program to investigate new and emerging diseases, identify environmental and management factors that influence the onset and severity of disease outbreaks; and. 5)establish the epidemiology of economically significant diseases, particularly those of unknown origin. 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? Activities of the Aquatic Diagnostic Laboratory, including case summaries and disease trends are and made available to producers, university and government officials in the form of an annual report. Research activities are described in a variety of formats, including publications of the Southern Regional Aquaculture Center, National Warmwater Aquaculture Center, a producer’s trade journal, and in scientific journals. Verbal transfer of information occurs through phone and laboratory consultations, and through various workshops, demonstration projects, on-farm visits, and at scientific meetings. Research associated with catfish health research has lead to 5 published abstracts in the proceedings of regional, national and international scientific meetings, 4 popular articles, and 11 scientific articles published in the Journal of the World Aquaculture Society, Aquaculture, American Journal of Aquaculture, Journal of the American Veterinary Medical Assoication, Veterinary Journal, Journal of Developmental and Comparative Immunology, and Journal of Veterinary Diagnostic Investigations. Specific projects that have generated Information and practical solutions to fish health issues are development of trematode management programs, identification and characterization of the toxin that causes visceral toxicosis of catfish (VTC), presentation of possible implications of VTC to industry and state agencies, and the development and implementation of management programs for the control bacterial infections using medicated feeds, vaccines and restricted feeding practices. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Popular Articles: Bilodeau, A.L., Small, B.C., Wolters, W.R., and Wise, D. J. 2005. Early host response improves disease resistance in channel catfish. Global Aquaculture Advocate. 8(3):84. Gaunt, P. and Schnick, R. 2005. Industry gathers to discuss aquaculture drug status. The Catfish Jounal. 20(2):21. Hanson, T. R. and Wise, D. J. 2005. Economic analysis projects 10% loss to Bolbophourus trematode in U. S. channel catfish industry. Global Aquaculture Advocate. 8(6):64. Camus, A. C., Guant P., Mauel, M. J. 2006. 2005 CVM Aquatic Diagnostic Laboratory Summary. NWAC News. 9(1):6-7. Steeby, J., Wise, D., Byars, T.,Thompson, L. 2006. Use of smallmouth buffalo (Ictiobus bubalus) to reduce the incidence of proliferative gill disease. NWAC News. 9(1):3. Wise, D., Hanson, T., Byars, T. 2006. Economic impact of trematode infections in commercially raised channel catfish. NWAC News. 9(1):4-5. Presentations: Wise, D. J. 2005. NWAC Fall Seminar. 2005. The effect of trematode infections on production of catfish. November 10, 2005. Delta Research and Extension Center, Stoneville, Mississippi. Wise, D. J. 2005. NWAC Fall Seminar 2005. The economic impact of trematode infections on production of catfish. December 2, 2005. , Mississippi State University, Starkville, Mississippi. Gaunt, P. 2006. Overview of florfeniol (Aquaflor®) research in channel catfish: Efficacy, safety, residue depletion studies, and implication for the catfish industry. Catfish Marketing Association Meeting, April 4, 2006, Greensboro, AL. Thompson, D. J., Khoo, L. H., Wise, D. J., and Hanson, L. A. 2006. Epidemiology of channel catfish virus disease. WAS International Symposium on Aquatic Animal Health. September 2-6, 2006. Wise, D. J. 2006. Texas Aquaculture Association. Impact of trematode infections on channel catfish. Jan 25-27, 2006, Bay City, Texas. Wise, D. J. 2006. Vaccination and integrated fish health management. American Chapter of the World Aquaculture Society. Feb 16-19, 2006. Las Vegas, Nevada. Wise, D. J. 2006. Producers meeting on the use of vaccines in an integrated fish health management program. Tribbet, MS. March 18, 2006.