2008 Annual Report 1a.Objectives (from AD-416) Updated objectives - covers the same scope as the original project: 1) Characterize the mechanisms of immune responses by in vitro and in vivo experiments to identify antibody and or cells responsible for protection;. 2)Develop and test non-living and modified live monovalent and multivalent vaccines and antibodies for passive immunization against economically important pathogens such as Lactococcus garviae, Streptococcus iniae, and S. agalactiae;. 3)Develop in ovo, bath hyperosmotic immersion, and oral routes of vaccine administration as a cost-effective means of mass immunization of fish with cohabitation and immersion vaccine evaluation as a means to more closely mimic field conditions. 1b.Approach (from AD-416) 1) In vitro and in vivo models will be developed to study the mechanisms of immune responses that will include passive immunization, antibody and cell-mediated assays, western blotting and protein electrophoresis of protective antigens;. 2)Vaccine master seeds will be identified by their virulence characteristics in tissue culture, organ cultures and/or fish experiments. Technologies used to develop domestic animal and human vaccines will be used to formulate and produce fish vaccines;. 3)Immersion and oral immunization protocols will be assessed to determine mass immunization strategies that will include encapsulation of vaccine into algal products and similar substances; and. 4)The influences of water temperature, salinity, dissolved oxygen, confinement and handling stress on the immunity responses following vaccination will be assessed in experimental vaccine trials and under field conditions. 3.Progress Report Bacterial and parasitic pathogens are responsible for considerable annual economic losses in channel catfish and tilapia. We worked to solve this problem by the development and use of biologics to prevent these important diseases. In addition, immunology, molecular biology and genomic approaches were important to development of vaccines. The projects that were done this year include: assessment of antibody and cellular immune responses against major fish pathogens; development of monovalent and multivalent non-living vaccines to prevent major fish diseases; development of multivalent bacterial vaccines to prevent major fish diseases; development of anti-parasite vaccines to prevent white spot disease of fish and development of cost-effective hyperosmotic and immersion vaccination strategies. The AQUAVAC-COL vaccine, Agricultural Research Service (ARS) licensed to an U.S. biologics manufacturer,is being evaluated for the prevention of columnaris disease in largemouth bass, trout and tilapia. Immunized largemouth bass were found to be protected. In trout field trials, the results suggest that the vaccine may have afforded protection. A Flavobacterium columnare challenge isolate was identified and successfully tested in tilapia. The objective of this research is to extend the license granted by ARS to an U.S. biologics manufacturer for the ARS invented vaccine to other species of fish susceptible to columaris disease. Research is being done under a CRADA with an U.S. biologics manufacturer to develop multivalent vaccines against Streptococcus iniae and S. agalactiae. Several trials were performed by both injection and bath immersion using bivalent vaccines in tilapia. Immunization by injection produced better survival than by bath immersion. However, bath immersion vaccination is a more cost-effective strategy of mass vaccination. Further experiments are being performed to improve the efficacy of bath immersion vaccination. Antibodies against F. columnare, S. iniae and S. agalactiae were found to provide the mechanisms of protection against these pathogens. Macrophage chemotaxis to extracellular products from E. tarda cells was demonstrated. The results demonstrated that the secretion of extracellular products by invading Edwardsiella tarda cells plays an important role in the initiation of inflammatory responses. The inflammatory responses of macrophages in turn are responsible for the initiation of immune responses of macrophages that play a role in protective immunity. Tilapia immunizations were done with live Ich, theronts or sonicated trophonts by bath immersion and injection. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. NP 106, Components: 4, Problem Statement: b, c, d, f. 4.Accomplishments 1. Assessment of antibody responses against major fish pathogens. Columnaris disease, caused by the bacteria Flavobacterium columnare, affects more than 36 fish species including channel catfish raised in the U.S. Vaccination provides immune protection in catfish against columnaris disease. The problems are what are the protective antigens and the mechanism of immunity that provide this protection? To solve these problems, we produced antibodies against two different antigen groups, lipopolysaccharide and killed whole cells in catfish. The antibodies revealed that lipopolysaccharide was an important protective antigen. The study demonstrated that specific antibody response is the mechanism of protective immunity against columnaris disease. NP 106, Components: 4, Problem Statement: c, d. 2. Assessment of cellular responses against major fish pathogens. The fish pathogen, Edwardsiella tarda, is the cause of an economically important disease in many species of cultured fish. The role of immunity is poorly understood and this has delayed the development of efficacious vaccines. Studies demonstrated that macrophages exposed to extracellular products secreted by E. tarda caused chemotaxis or movement of macrophages toward the extracellular products. The study demonstrated that extracellular products secreted from invading E. tarda cells play a role in the initiation of the inflammatory responses. NP 106, Components: 4, Problem Statement: c, d. 3. Anti-parasite vaccine to prevent white spot disease in fish. Ichthyopthirius multifiliis (Ich) is an economically important parasite and no vaccines are currently available. The Ich immobilization antigen has been isolated from Ich theronts electrophoresis and western blots. The gene encoding the antigen was sequenced. Immunizations were done with live theronts or sonicated trophonts by bath immersion or injection. Host protection was acquired in fish immunized with live theronts by immersion or injection. Tilapia immunized with sonicated trophonts were partially protected. The estimated savings by using an Ich vaccine will be significant to cultured fish and tropical fish producers. NP 106, Components: 4, Problem Statement: b, c, f. 4. Development of multivalent bacterial vaccines to prevent major fish diseases. No vaccine is currently available for the bacterial pathogen, Aeromonas hydrophila. Streptococcus iniae and A. hydrophila are often concurrent infections in tilapia. A modified live A. hydrophila vaccine was produced and shown to be effective both in channel catfish and tilapia in preventing Aeromonas disease. Successful prevention of streptococcal and Aeromonas diseases with a bivalent vaccine will increase the survival of cultured fish. The AQUAVAC-COL (ARS licensed to an U.S. biologics manufacturer) vaccine is being evaluated for the prevention of columnaris disease in largemouth bass, trout and tilapia. In trout field trials, results demonstrated that the vaccine may have afforded protection. Results in largemouth bass showed that this vaccine was efficacious against columnaris. Research is being done under a Cooperative Research and Development Agreement (CRADA) with an U.S. biologics manufacturer. NP 106, Components: 4, Problem Statement: b, f. 5. Development of monovalent and multivalent non-living vaccines to prevent major fish diseases. Streptococcal diseases are significant problems worldwide. Streptococcal pathogens cause hemorrhage and septicemia in tilapia, hybrid striped bass, rainbow trout, olive flounder, yellowtail, barramundi and other species of cultured and wild fish. Research is being done under a CRADA with an U.S. biologics manufacturer to develop multivalent vaccines against Streptococcus iniae and S. agalactiae. A bivalent S. iniae and S. agalactiae vaccine was produced and tested in experimental trials. The results of experimental trials show that injection of tilapia produced good survival against both of these pathogens. Concurrent streptococcal infections are known to occur in tilapia production and a bivalent vaccine would be very valuable to producers in preventing these infections. The estimated savings of a bivalent streptococcal vaccine would be significant to the prevention of two major streptococcal infections with a single vaccine application. NP 106, Components: 4, Problem Statement: b, c. 6. Develop cost-effective hyperosmotic and immersion immunization vaccination. Injection vaccination is not a cost-effective method for mass immunization of warm-water fishes, like tilapia. The problem is a lack of cost-effective mass vaccination strategies. One solution was to perform a primary bath immunization at the age of immunocompetency followed by two bath immunizations at 30 to 60 day intervals. The bath immersion immunization was performed using a killed whole cell S. iniae vaccine supplemented with extracellular products. Immunized fish were protected against S. iniae disease. It was concluded that the mechanism of protective immunity was due to the antibody response following immunization. The development of a bath immersion immunization strategy will have added benefits to the costs associated with immunization and losses from disease. NP 106, Components: 4, Problem Statement: b, c. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Active CRADAs 1 Number of the New MTA (providing only) 1 Number of New Commercial Licenses Executed 4 Number of Other Technology Transfer 1 Review Publications Klesius, P.H., Shoemaker, C.A., Evans, J.J. 2008. Bath immersion, booster vaccination strategy holds potential for protecting juvenile tilapia against Streptococcus iniae. Global Aquaculture Advocate. 11(3): 49-50. Abernathy, J.W., Xu, P., Li, P., Xu, D., Kucuktas, H., Klesius, P.H., Arias, C., Liu, Z. 2007. Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis. Biomed Central (BMC) Genomics. 8:176 pgs. 1-10. Xu, D., Klesius, P.H., Shoemaker, C.A. 2008. Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post immunization with live theronts and sonicated trophonts. Fish and Shellfish Immunology. 25: 124-127. Wiedenmayer, A.A., Klesius, P.H., Evans, J.J., Shoemaker, C.A. 2008. The macrophage chemotactic activity of Edwardsiella tarda extracellular products. Journal of Fish Diseases. 31(5):331-342. Klesius, P.H., Evans, J.J., Shoemaker, C.A. 2007. Immunostimulation, vaccine and phage therapy strategies in aquaculture. In: Aquaculture Health International, Issue 11: 36-38. Shelby, R.A., Shoemaker, C.A., Klesius, P.H. 2007. Passive immunization of channel catfish Ictalurus punctatus with anti-Flavobacterium columnare sera. Diseases of Aquatic Organisms. 77: 143-147.