2007 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) 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 None. 4.Accomplishments 1. Assess antibody and cellular immune responses against major fish pathogens. Serum and mucus antibody responses that may be involved in protective effects against Streptococcus iniae, S. agalactiae and Ichthyophthirius multifillis were assessed. Passive transfer with serum antibody from S. iniae and S. agalactiae immune tilapia protected tilapia from infection with S. iniae and S. agalactiae, respectively. Mucus antibody from the skin of channel catfish immune to Ichthyophthirius immobilized theronts of Ich and prevented Ich infections. Research demonstrated that extracellular products of the streptococcal vaccines are important pro-inflammatory molecules that initiate the cellular immune response to streptococcal infections and vaccination. Knowledge of antibody and cellular immune responses is essential for the development of vaccines against major fish pathogens. NP 106 (Action plan components IIc-Immunology and disease resistance and IId- Mechanisms of disease). 2. Development of monovalent and multivalent non-living vaccines to prevent major fish diseases. Streptococcal disease results in more than $200 million dollars annually in fish and streptococcal vaccines are not available. Vaccines to control the bacterial pathogens, S. agalactiae and S. iniae, were developed and patented. The vaccines do not leave a residue, so are safe for the fish and the consumer. Successful prevention of fish streptococcal disease with these vaccines will increase the viability and productivity of important fish species. The vaccines are being licensed by Agricultural Research Service(ARS) Office of Technology Transfer (OTT) to a major vaccine manufacturer for use as a bivalent vaccine in the USA and worldwide. The expected economic impact of the vaccines is $100 million dollars annually. NP 106 (Action plan components IIb- Vaccines and medicine and 4f-Microbial genomics). 3. Develop multivalent bacterial attenuated vaccines to prevent major fish diseases. No vaccines were available to prevent enteric septicemia of catfish and columnaris, the two major bacterial diseases of catfish. Modified live immersion vaccines were developed under a Cooperative Research and Development Agreement (CRADA) with a major vaccine manufacturer, patented and exclusively licensed to the vaccine manufacturer. The patent/license transfer mechanism used was essential to the successful commercialization of these vaccines. According to the vaccine manufacturer, total benefit to producers from use of these vaccines is almost $2,000 per acre because the faster growing vaccinated catfish are ready for market sooner than unvaccinated catfish. These two vaccines, in combination, provide fish farmers a cost effective means for preventing the two most economically serious diseases affecting commercial pond-raised catfish. Currently, fry production is about 1 billion/year and some 25% are vaccinated with one or both of the vaccines. With some 180,000 acres of ponds in catfish production, the potential economic benefit of these vaccines approaches $50 million annually. NP 106(Action plan components IIb- Vaccines and medicine and 4f-Microbial genomics). 4. Anti-parasite vaccine to prevent white spot disease of fish. Ichthyopthirius multifiliis (Ich) is a major parasitic fish pathogen and no vaccines are currently available. Molecular investigations, which are in progress, will determine the nature of the protective Ich antigens and aid in the development of an efficacious vaccine. Catfish were protected against Ichthyopthiriosis following bath immersion immunization with live theronts or by injection with sonicated trophonts. Thus, cutaneous antibody plays an important role in protecting against Ich including those produced in the skin. The development of a novel vaccine against Ich is expected and its impact will be of considerable economic benefit to fish producers. The estimated saving of an Ich vaccination may exceed $10 million dollars annually. NP 106(Action plan components IIb- Vaccines and medicine and 4f-Microbial genomics). 5. Develop cost effective hyperosmotic and immersion immunization vaccination methods. Streptococcal diseases are responsible for economic losses of more than $150 million dollars annually and injection vaccination is not a cost effective method for vaccination of warm-water fishes, like tilapia. Streptococcus iniae and S. agalactiae vaccines may be administered to tilapia weighing between 0.01 to 25 grams or more by hyperosmotic immersion. The benefits of the immersion vaccination technique outweigh the costs of vaccine and immunization. Immunization may profit the farmer by $100 - $300 more per acre because fish may eat more food, grow faster and suffer less mortality. Another major vaccine manufacturer is cooperating by a CRADA to commercialize a combination vaccine against S. iniae and S. agalactiae for a world-wide market. This combination vaccine is expected to be available in 2008 and may provide greater than 90% survival against both pathogens. The estimated saving is $80 million dollars annually, worldwide. NP 106(Action plan components IIb- Vaccines and medicine and IIg- Aquatic animal health management). 5.Significant Activities that Support Special Target Populations On-farm efficacy and cost-effectiveness of columnaris vaccine. A commercial columnaris vaccine that was developed at this laboratory has been licensed for use in catfish. However, efficacy of this vaccine when used on the rainbow trout farm has not been determined. In March, 2007, this vaccine was administered to brook and brown trout fry at a North Carolina farm that experiences annual columnaris outbreaks. The information obtained from this study will help broaden the application of this product to other aquaculture species. Field trial data relating to the efficacy of the commercial columnaris vaccine that was developed at this laboratory is being collected at large farms that produce catfish fingerlings. However, small farms (i.e., less than 20 water acres) may also benefit from the use of this product. In May/June, 2007, the vaccine was administered to fry at a small Alabama catfish farm. Field data describing the benefits and costs of the use of this vaccine will provide valuable information about the range of farm types that could profit from its use. 6.Technology Transfer Number of new CRADAs and MTAs 2 Number of active CRADAs and MTAs 1 Number of patent granted 1 Number of U.S. patents granted 1 Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 7 Number of newspaper articles and other presentations for non-science audiences 3 Review Publications Evans, J.J., Klesius, P.H., Shoemaker, C.A. 2006. An overview Streptococcus in warm-water fish. Aquaculture Health International. Issue 7 pg. 10-14. Klesius, P.H., Evans, J.J., Shoemaker, C.A. 2007. The macrophage chemotactic activity of Streptococcus agalactiae and Streptococcus iniae extracellular products (ECP). Fish and Shellfish Immunology. 22(5)443-450. Klesius, P.H., Evans, J.J., Shoemaker, C.A., Lim, C.E. 2007. A US perspective on selected biotechnological advancements in fish health Part II: Genetic stock improvement, biosecurity tools and alternative protein sources in fish diets. Aquaculture Health International. Issue 9:18-20. Xu, D., Klesius, P.H., Shoemaker, C.A. 2007. Evaluation of a cohabitation challenge model in immunization trials for channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis. Diseases of Aquatic Organisms. 74: 49-57. Shoemaker, C.A., Klesius, P.H., Evans, J.J. 2006. Immunization of eyed channel catfish, Ictalurus punctatus, eggs with monovalent Flavobacterium columnare vaccine and bivalent F. columnare and Edwardsiella ictaluri vaccine. Vaccine. 25(2007):1126-1131. Klesius, P.H., Shoemaker, C.A., Evans, J.J. 2007. Tilapia Vaccines: Important Disease Prevention, Biosecurity Tools. Global Aquaculture Advocate May/June page 76, 78. Klesius, P.H., Evans, J.J., Shoemaker, C.A., Pasnik, D.J. 2006. Streptococcal vaccinology in aquaculture. Book Chapter in Tilapia Biology, Culture, and Nutrition. Chhorn E. Lim and Carl D. Webster editors. Food Products Press an imprint of The Haworth Press Inc.,Binghamton, New York. Chapter 17 p. 583-606.