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? 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 diseases may exceed 10% of the estimated farm gate value of S1.3 B and this cost is likely to increase in more intense production systems. In addition, the negative environmental and food 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 US. 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 the milestones (indicators of progress) from your Project Plan. 1) Assess antibody response. 2) Assess cellular response. 3) Develop monovalent and multivalent bacterial non-living vaccines. 4) Develop multivalent bacterial attenuated living vaccines. 5) Develop monovalent anti-parasitic (Ich) non-living vaccine. 6) Assess in ovo immunization. 7) Assess hyperosmotic immersion immunization. 8) Assess OralJect feed immunization method. 9) Assess spray dried vaccine preparations for oral or feed delivery. 10)Assess cohabitation and immersion challenge method. 4a.What was the single most significant accomplishment this past year? Flavobacterium columnare attenuated vaccine. Columnaris disease is among the top two diseases of catfish and is estimated to cost the producer $50 million annually. An attenuated live vaccine was developed, patented (US Patent No. 6,881,412)and licensed to Intervet for protecting catfish against columnaris disease. The vaccine is licensed to be used in catfish at 7-10 days post-hatch and is given by bath immersion. Thus, large numbers of catfish (10,000 or more) can be immunized by a 2 minute exposure to the vaccine in the hatchery with minimal or no stress. The immunized fish are protected against F. columnare infection for their full production cycle. The economic impacts are catfish that yield more weight, grow faster and have significantly less mortality due to this disease than non-immunized catfish. The estimated profit to the producer is $60 million, annually. 4b.List other significant accomplishments, if any. 1. Streptococcus agalactiae killed vaccine. A non-living vaccine against S. agalactiae was developed and experimentally evaluated for its safety and efficacy. The vaccine can be administered by injection or immersion. The ELISA antibody response to S. agalactiae extracellular product and protection against S. agalactiae following vaccination was successfully correlated. The protective antigen of the extracellular product was identified by western blot analysis using immune serum. The vaccine efficacy was shown to have a duration of 6 months following a single vaccination by injection. A patent was filed on this S. agalactiae vaccine and it is pending a U.S. patent office approval. 2. Oral immunization. In an agreement between ARS and PerOs Systems Technologies, Inc., the patented S. iniae vaccine was formulated in the PerOs, OralJect feed vaccine and tested for its efficacy in tilapia. The results showed that tilapia fed OralJect 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. 3. Cohabitation and immersion challenge model. Fish were non invasively and safely chemically marked and cohabitated with non-marked fish. The chemically marked fish were immunized and non-marked sham immunized. Marked and non marked fish were challenged at the same time in the same aquarium and the efficacy of the vaccine determined by identifying marked and non-marked fish. The development of this cohabitation and immersion challenge model stimulated more natural conditions (i.e.in production facilities) and more statistical power (i.e. each fish is a experimental unit)to assess vaccine efficacy. 4. Ich immunodominant antigen. Immunoaffinity chromography and western blot analysis immunodominant antigens of Ich trophonts were identified. Also, antigen specific for Ich theront, trophont and tomont life stages were also identified using SDS-PAGE and western blot analysis. No further disclosures of the immunodominant 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 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. 5.Describe the major accomplishments over the life of the project, including 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 food 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 vaccine demonstrated exceptional creativity in the invention and transfer of the first U.S, modified live-vaccines that 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. ictalari, 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. The modified live vaccines are administered, by bath immersion, a non-stressful and inexpensive process, to large numbers of young fish and provides 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 2007 and licensed by FY 2008. 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 Intervet, Inc., 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, 2005), and exclusively licensed to Intervet. The CRADA allowed Team members to develop the final freeze dried formulations in cooperation with Intervet scientists and to cooperatively test the vaccines’ safety and effectiveness. 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 (AQUAVAC-ESCTM) was first introduced in 2001. According to Intervet, total benefit to producers from use of this vaccine alone is almost $2,000 per acre, due to faster growing catfish that yield greater lengths over non-vaccinated catfish. Since AQUAVAC-ESC’s release almost 1 billion fry have been vaccinated. The columnaris vaccine (AQUAVAC-COLTM), the first efficacious vaccine against columnaris disease in the world, was launched in 2005, and the 2005 production is 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. AQUAVAC-ESC and AQUAVAC-COL are both administered via bath immersion at 7 days post hatch to prevent disease losses of $50 million or more annually. Other benefits of use of these vaccines include increased catfish yields, due to improved growth and survival. Use of these vaccines significantly reduces 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. 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). USDA, ARS, Agricultural Research, Protecting Fish Through Vaccines. May, 2005. P. 10-11. Klesius, P.H., Evans, J.J., Shoemaker, C.A. Warm water fish vaccinology in catfish production. Animal Health Research Reviews. 2004. 16(3/4):305-311. Klesius, P.H., Evans, J.J., Lim C., Pasnik, D.J. Aquaculture 2005 presentation for Nutrition and Fish Health Special Session, entitled “Dietary strategies to improve vaccination efficiency”. New Orleans. January. Yu, X.L., Gan, X. Liang, W.W., Xu, D.H. Current research on fish parasite Ichthyophthirius multifiliis. Journal of Modern Fisheries Information. 19:14-17. Shoemaker, C.A., Klesius, P. H., Evans, J. J. Modified live Flavobacterium columnare vaccine against columnaris disease in fish. 2005. U.S. Patent 6,881,412 B.1. Review Publications Evans, J.J., Klesius, P.H., Shoemaker, C.A. 2004. Efficacy of streptococcus agalactiae (group B) vaccine in tilapia (oreochromis niloticus) by intraperitoneal and bath immersion administration. Vaccine. vol 22 (27-28). p. 3769-3773. EVANS, J.J., KLESIUS, P.H., SHOEMAKER, C.A., FITZPATRICK, B.T. STREPTOCOCCUS AGALACTIAE VACCINATION AND INFECTION STRESS IN NILE TILAPIA, OREOCHROMIS NILOTICUS. JOURNAL OF APPLIED AQUACULTURE. 2004. vol 16 numbers 3/4:105-115. Klesius, P.H., Evans, J.J., Shoemaker, C.A., Pasnik, D.J. 2006. A vaccination and challenge model using calcein marked fish. Fish and Shellfish Immunology 20: 20-28. Pasnik, D.J., Evans, J.J., Panangala, V.S., Klesius, P.H., Shelby, R.A., Shoemaker, C.A. 2005. Antigenicity of streptococcus agalactiae extracellular products and vaccine efficacy. Journal of Fish Diseases. 28(4):205-212. Bader, J.A., Shoemaker, C.A., Klesius, P.H. 2005. Production, characterization and evaluation of virulence of an adhesion defective mutant of flavobacterium columnare produced by b-lactum selection. Letters in Applied Microbiology. Vol. 40: 123-127. Shoemaker, C.A., Xu, D., Shelby, R.A., Klesius, P.H. 2005. Detection of cutaneous antibodies against flavobacterium columnare in channel catfish, ictalurus punctatus, (rafinesque). Aquaculture Research 36(8): 813-818.