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? Practical information on immune system function of warm water fish, especially on the antibody or cellular immune response of fish following vaccination and infection, is very limited. The immediate need to develop safe and effective vaccines is critical to the well being of aquaculture. Diseases of cultured fish cause losses of hundreds of million dollars annually. The cost of catfish and tilapia disease may exceed 10% of the estimated farm gate value of $1.3 billion dollars and this cost is likely to increase in more intense production systems. In addition, the negative environmental and feed safety issues associated with the use of chemotherapeutics are avoided by the use of vaccines in integrated aquatic animal health management. These chemotherapeutic issues have contributed to a ban on sub therapeutic antibiotic usage in Europe and a possible ban in the U.S. Emergence and re-emergence of pathogens is an often overlooked problem that vaccination can solve. Thus, the goals of this project are to assess immune mechanisms responsible for protection against pathogens and following vaccination; develop fish vaccines to prevent diseases caused by economically important pathogens; create and test mass vaccination strategies and to evaluate and assess husbandry practices and environmental conditions that may cause vaccine failure. 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. 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. 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. The influences of water temperature, salinity, dissolved oxygen, confinement and handling stress on the immune responses following vaccination will be assessed in experimental vaccine trials and under field conditions. New and improved vaccines will be available to prevent diseases and reduce dependency on chemotherapeutics, thus the consumer, public and producer will significantly benefit from the outcomes of this project. 2.List by year the currently approved milestones (indicators of research progress) Milestone 1. 2004-2007- Assess antibody response. Milestone 1.2. 2004-2007- Assess cellular response. Milestone 2.1 2004-2007- Develop monovalent and multivalent bacterial non-living vaccines. Milestone 2.2 2004-2008- Develop multivalent bacterial attenuated living vaccines. Milestone 2.3 2004-2007- Develop monovalent anti-parasitic (Ich) non-living vaccine. Milestone 3.1 2004-2008- Assess in ovo immunization. Milestone 3.2 2004-2008- Assess hyperosmotic immersion immunization. Milestone 3.3 2004-2008- Assess OralJect feed immunization method. Milestone 3.4 2004-2008- Assess spray dried vaccine preparations for oral or feed delivery. Milestone 3.5 2004-2008- Assess cohabitation and immersion challenge method. 4a.List the single most significant research accomplishment during FY 2006. Streptococcal vaccines AAHRU scientists have developed novel vaccines to control the bacterial pathogen, Streptococcus agalactiae and S. iniae, in fish. S. agalactiae and S. iniae have been associated with significant mortalities among a variety of freshwater, estuarine, and marine fish species. Streptococcal disease results in more than $100 million in losses annually in cultured fish. Currently, no approved antibiotics are available to treat the pathogen because they are hazardous to the environment. The streptococcal vaccines protect fish against multiple pathogen strains of these species. The vaccines consist of formalin-killed cells and concentrated ex-cellular products from the streptococcal broth culture. It can be administered to fish weighing 0.5 to 10 grams or more by injection or bath immersion. The S. agalactiae vaccine was shown to provide a theurapeutic effect in sub clinically infected tilapia by protecting these fish from further morbidity and mortality due to S. agalactiae. The vaccines do not leave a residue, therefore are both safe to the fish and persons eating the fish. Successful prevention of fish streptococcal disease with these vaccines will increase the viability and productivity of important fish species. The benefits of the immersion vaccination technique out weigh 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. A major pharmaceutical manufacturer is cooperating by a Cooperative Research and Development Agreement (CRADA) to commercialize these vaccines for a world-wide market. 4b.List other significant research accomplishment(s), if any. 1. Oral immunization In an agreement between Agricultural Research Service (ARS) and a company working on vaccine delivery systems, the patented S. iniae vaccine was formulated in the feed vaccine and tested for its efficacy in tilapia. The results have shown that tilapia fed vaccine were protected against experimental challenge with S. iniae. The concept of using an oral vaccine feed appears to be a promising mass immunization method to protect fish against disease, but additional research is needed to make this concept work under farm conditions. 2. Monovalent attenulated living vaccine An attenuated vaccine against Edwardsiella tarda was developed and patented (U.S. Patent #7,067,122) in 2006. Edwardsiellosis is a serious disease of many species of cultured fish including catfish. Economic losses may exceed $70 millon (M) annually, world-wide. This attenuated vaccine is given by bath immersion to catfish and tilapia at an early age. A vaccine manufacture working to further develop this vaccine for the cultured fish market. The benefit of using this vaccine may be expected to exceed $25 M annually. 3. Cellular immune mechanisms Macrophage stimulating activity of S. iniae and S. agalactiae ex-cellular product vaccines was characterized to help better elucidate immune mechanisms. This research demonstrated that ex-cellular products are important pro-inflammatory molecules that help recruit macrophages and initiate the cellular immune response to streptococcal infections and vaccination. 4. Immune antibody mechanisms Passive immunization with antibody from S. agalactiae immune fish was demonstrated to protect susceptible fish to challenge infection with S. agalactiae. These findings demonstrate that immune antibody plays a role in protective immunity in fish immunized with the ARS S. agalactiae vaccine. 5. Ich immunodominant antigen Immunoaffinity chromography and western blot analysis of immunodominant antigens of Ich trophonts were identified. Also, antigen specific for Ich theront, trophont and tomont life stages were identified using SDS-PAGE and western blot analysis. No further disclosures of the antigen can be made because of patentability issues on the development of an Ich vaccine. The Ich antibodies initiated apoptosis of Ich theronts and thus were responsible for protective immunity against Ich, at least in part. These antibodies are responsible for neutralization of theronts by inhibiting their ability to move and to penetrate skin of the host fish. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. The project will directly benefit the goals, approaches and outcomes of the Aquaculture 106 Action Plan. The goals are to develop safe and effective vaccines and medicines for prevention and control of economically important pathogens of aquatic animals; identify effective mass delivery strategies for aquatic animal vaccines; conduct research and development to support approval and licensing of new aquatic animal vaccines. The approaches to meet these goals are produce vaccines that can be experimentally tested in the laboratory for safety and effectiveness; apply vaccines using mass delivery strategies in on-farm trials; and evaluate the safety and effectiveness of new aquatic animal vaccines in approved trials to aid the licensing agencies (i.e., USDA-APHIS-CVB, FDA). The expected outcomes are: (1) new killed, modified live, DNA and recombinant vaccines; (2) methods for mass vaccination will be developed; and (3) environmentally friendly, effective and safe vaccines to treat aquatic animal diseases will become available. The project entitled "Vaccinology and Immunity of Aquatic Animals" was certified by the Office of Scientific Quality in May, 2005. The attenuated live vaccines demonstrated exceptional creativity in their invention and transfer to industry. These vaccines protect channel catfish from enteric septicemia and columnaris. Enteric septicemia and columnaris are the two major diseases of U.S. farm raised catfish and were reported to be a problem on about 50% of all catfish operations in 2003. Edwardsiella septicemia caused by E. tarda is also among the major disease problems of catfish. Diseases together cost the U.S. catfish industry $50-70 million annually. The causative bacteria, E. ictaluri, F. columnare and E. tarda, respectively, are ubiquitous pathogens that infect all sizes of catfish. No effective control measures were available to the farmer. These pathogens are responsible for severe disease outbreaks throughout the catfish industry every year. Previous studies suggested that killed vaccines against E. ictaluri, F. columnare and E. tarda were not effective when administered by immersion (i.e. mass delivered in the field). ARS modified these agents with an antibiotic that changed the lipopolysaccharide (a virulence factor of Gram-negative bacteria). The modified bacteria were still able to gain entry into the fish for a proper immune response to develop but could no longer cause disease. By creation of the modified live vaccines, the problems associated with killed vaccines (e.g. injection administration of each fish, cost of administration, stress associated with administration, and not providing lifetime protection) were overcome. These modified live vaccines are administered by bath immersion, a non-stressful and inexpensive process to large numbers of young fish and provide life long protection. These modified live vaccines against E. ictaluri, F. columnare and E. tarda are possibly a trend setting advancement for the rest of the world in fish vaccinology. Vaccines against Streptococcus iniae, S. agalactiae, L. garviae and A. hydrophila are expected to be available in FY 2008 and licensed by FY 2007. Mass immunization methods are also expected to be available in FY 2007 and FY 2008. 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? Both vaccines were developed under a Cooperative Research and Development Agreement (CRADA) with a major vaccine manufacturer, patented (U.S. Patent No. 6,019,981, Modified Live E. ictaluri Against Enteric Septicemia in Channel Catfish, 2002 and U. S. Patent No. 6,881-412, Modified Live F. columnare Against Columnaris Disease in Fish, 2006), and exclusively licensed to the vaccine manufacturer. The CRADA allowed team members to develop the final freeze dried formulations in cooperation with manufacturer's scientists and to cooperatively test the vaccines' safety and effectiveness. ARS and the vaccine company are titrating optimal immunodoses of the frozen product ESC vaccine that will provide efficacy results in 7 day-post hatch catfish in 2006-2007. The patent/license transfer mechanism used was essential to the successful commercialization of these vaccines, in part, because of the costs associated with obtaining regulatory approval for their use. The enteric septicemia vaccine was first introduced in 2001. According to the vaccine manufacturer, total benefit to producers from use of this vaccine along is almost $2,000 per acre, due to faster growing catfish that yield greater lengths over non-vaccinated catfish. Since ESC vaccine release almost 1 billion fry have been vaccinated. The columnaris vaccine. the first efficacious vaccine against columnaris disease in the world was launched in 2005, and the 2005/2006 production was sold out. The columnaris vaccine is safe and provided 72% improved survivability of catfish fry in laboratory studies when challenged with the virulent bacteria. These vaccines, in combination, provide fish farmers a cost effective means for preventing the two most economically serious diseases in commercial pond-raised catfish. Both the ESC and columnaris vaccines are administered via bath immersion at 7 days post hatch to prevent disease losses. Other benefits of use of these vaccines include increased catfish yields, due to improved growth and survival. Use of these vaccines significantly reduced the need for antibiotics thus, decreasing environmental contamination and providing a safe fish product to consumers. 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. ARS vaccine master seeds of S. iniae and S. agalactiae isolates have been transferred to another vaccine company for the development of a Streptococcus iniae vaccine and an S. agalactiae vaccine or combination vaccine for use in warm-water fish species. The benefit of these streptococcal fish vaccines to the fish producers is the prevention of morbidity and mortality caused by streptococcal disease that costs the industry more than $150 M annually. 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). 1. USDA, ARS Agricultural Research. Protecting Fish through Vaccines. May, 2005. pp. 10-11. 2. Innovation: America Journal of Technology Commercialization. FLC's Tech transfer award Winners. P. 27-28. June/July 2006. 3. ARS Research News: ARS Scientists Honored for Tech Transfer. February, 2006. 4. The Catfish Journal: Scientists Recognized for Work on Vaccine for ESC in Catfish. March, 2006. 5. ARS Research News: ARS Scientists Honored for Catfish Research. June, 2006. 6. Tilapia Update. World Aquaculture. 37(2):19. Review Publications Klesius, P.H., Evans, J.J., Shoemaker, C.A. 2006. Advancements in fish vaccine development. Aquaculture Health International. Issue 4 February 2006 page 20-21. Klesius, P.H., Evans, J.J., Shoemaker, C.A., Lim, C.E. 2006. A US Perspective on Selected Biotechnological Advancements in Fish Health part 1: Vaccines. Aquaculture Health International. Issue 5 May 2006 pg 10-12 cont. on 21. Evans, J.J., Klesius, P.H., Shoemaker, C.A. 2006. Therapeutic and prophylactic immunization against streptococcus iniae infection in hybrid striped bass (morone chrysops x m. saxatilis). Aquaculture Research. 37:742-750. Pasnik, D.J., Evans, J.J., Klesius, P.H. 2006. Passive immunization of nile tilapia (oreochromis niloticus) provides significant protection against streptococcus agalactiae. Fish and Shellfish Immunology. Vol 21 Issue 4 pages 365-371. Xu, D., Klesius, P.H., Panangala, V.S. 2006. Induced cross protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes of Ichthyophthirius multifiliis. Journal of Fish Diseases, 29: 131-138. Xu, D., Klesius, P.H., Shoemaker, C.A. 2006. Apoptosis in Ichthyophthirius multifiliis is associated with expression of Fas receptor of theronts. Journal of Fish Diseases 29: 225-232. Shoemaker, C.A., Klesius, P.H., Evans, J.J. 2006. Method of protecting fish against columnaris disease with modified live Flavobacterium columnare. U.S. Patent US 6,991,793 B2. Klesius, P.H., Shoemaker, C.A., Evans, J.J. 2005. Attenuated vaccines against gram-negative bacterial pathogens. 6th Symposium on Diseases in Asian Aquaculture Proceedings. Colombo Plaza Hotel, Colombo, Sri Lanka. October 25-28, 2005. Klesius, P.H., Shoemaker, C.A., Evans, J.J. 2006. Modified live vaccines for gram negative pathogens of catfish. Aquaculture America Conference 2006. Las Vegas, Nevada. Febrauary 13-16, 2006. page 153. Evans, J.J., Klesius, P.H., Shoemaker, C.A. 2006. Therapeutic and prophylactic immunization against streptococcus iniae infection in hybrid striped bass morone chrysops x m. saxatilis. Aquaculture America Conference 2006. Las Vegas, Nevada. February 13-16, 2006. page 97. Xu, D., Klesius, P.H., Panangala, V.S. 2006. Ichthyophthirius (Ich) induced cross protection in channel catfish against different serotypes of Ich. Aquaculture America Conference. Klesius, P.H., Shoemaker, C.A., Evans, J.J. Attenuated vaccines for Edwardsiella ictaluri and Flavobacterium columnare of catfish. 2005. Research Workers in Animal Diseases Conference Proceedings, Special Session. St. Louis, MO. Shoemaker, C.A., Vandenberg, G.W., Desormeaux, A., Klesius, P.H., Evans, J.J. 2006. Efficacy of a Streptococcus iniae modified bacterin delivered using Oralject TM technology in Nile tilapia (Oreochromis niloticus). Aquaculture 255, 151-156. Shoemaker, C.A., Vandenberg, G., Desormeaux, A., Klesius, P.H., Evans, J.J. 2006. Delivery of a streptococcus iniae vaccine using oralject TM technology in tilapia oreochromis niloticus¿. Aquaculture America Conference 2006. Las Vegas, Nevada. February 13-16, 2006. page 279. Evans, J.J., Klesius, P.H., Shoemaker, C.A., Pasnik, D.J. 2006. Streptococcal vaccinology in warm-water fish. Aqua 2006 includes: World Aquaculture 2006 and Aquaculture/Europe2006. May 9-13, 2006. Florence, Italy. p. 293. Klesius, P.H., Evans, J.J., Shoemaker, C.A. 2006. Benefits of the use of attenuated vaccines in aquaculture. Aqua 2006: including World Aquaculture 2006 and Aquaculture Europe 2006. May 9-13, 2006. Florence, Italy. p.468. Xu, D., Klesius, P.H., Shoemaker, C.A. 2005. Cutaneous antibodies from catfish immune to Ichthyophthirius (Ich) induced apoptosis of Ich theronts. American Fisheries Society Annual Meeting. Page 386. Evans, J.J., Klesius, P.H., Shoemaker, C.A. 2006. A modified-live Edwardsiella tarda vaccine for aquatic animals. U.S. Patent 7,067,122.