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? Aquatic animal farmers in the U.S. continue to identify disease as a major problem in their industry. Loss to catfish farmers alone exceeds $60-80 million annually. An area of inadequate research is in rapid detection and identification of pathogens and off-flavor compounds. The appropriate methods and reagents to rapidly (within hours) detect pathogens (or toxins) and diagnose disease in aquatic species in a non-lethal manner are either not available or have not been applied at the farm or in the aquatic environment. Early detection will also allow for earlier intervention with preventative measures and help to reduce or eliminate the impact of emerging or catastrophic diseases in U.S. aquaculture and aquatic environments. There is also a lack of knowledge of the pathogenesis (i.e. mechanism of disease) of aquatic animal pathogens. Basic information is needed on the source of infection (i.e. water, wildlife, carrier fish, agriculture), modes of transmission (i.e. vertical from mother to offspring or horizontal from fish to fish), routes of entry (i.e. gills or nares), mechanisms of pathogen virulence, host immune response and the influences of toxic algae, water quality and other environmental factors on host immune system and disease transmission. The lack of adequate in vivo (i.e., in the fish) and in vitro (i.e., laboratory) models to investigate the mechanism of disease and virulence of pathogens is hindering our progress. Applied epidemiology studies are also lacking in aquatic animal production and aquatic ecosystems. One example is in catfish production where often producers cannot account for over 70% of their stocked animals at time of harvest. The overall goal of this research project is to provide fish farmers and fish health manager's tools for diagnosing and identifying pathogens and off-flavor compounds in aquaculture and to answer basic questions on disease transmission and epidemiology. The approaches include development and testing of the specificity and sensitivity of monoclonal and polyclonal antibody assays, molecular probes (Polymerase Chain Reaction (PCR)) and test systems focused on rapid non-lethal detection and identification of pathogens. Off-flavor compound detection will employ dogs (canine olfactory detection) and will focus on water samples from ponds and fish fillets. Basic studies on the mechanism of disease will be set up to address both horizontal (i.e., fish to fish) and vertical (i.e., mother to off-spring) transmission of fish pathogens. The final approach will consist of multidisciplinary research using microbiology, parasitology, immunology, histology and nutrition to answer basic questions regarding the epidemiology of fish pathogens. Meeting the objectives of this multifaceted project will provide information that can be used by fish farmers and aquatic animal health managers to help reduce the economic impact of health problems in aquatic animal production. Regulatory agencies and biotechnology companies may benefit from the reagents and potential kits developed to detect and/or identify the major aquatic animal pathogens. 2.List the milestones (indicators of progress) from your Project Plan. The milestones to be addressed include: . 1)production of monoclonal and polyclonal antibodies, nucleic acid sequencing and primer design for polymerase chain reaction assays for development of rapid detection methods for fish pathogens; . 2)field testing of dogs for the detection of off-flavor compounds;. 3)development of in vitro models to assess monogenetic trematode infections;. 4)investigate water borne transmission of F. columnare in channel catfish and characterize the effect of fish density and bacterial dose on F. columnare infection in channel catfish. (5) The milestones for the epidemiology studies include the development of protocols and methods to investigate F. columnare/E. ictaluri presence in channel catfish and to begin to select sites and develop protocols for the study of proliferative gill disease; and. 6)conduct studies to assess the relationship between harmful algae, stress and bacterial infection in the Cheasapeake Bay system. 4a.What was the single most significant accomplishment this past year? The major accomplishment of this project was the transfer of dogs with the ability to detect off-flavor compounds to the Alabama Fish Farming Center in Greensboro, AL to determine the potential utility of canine detection of off-flavor in the field. Off-flavor (an earthy or musty taste) in catfish production costs the industry as much as $50 million annually. Dogs selected from a local animal shelter were trained to sniff water samples for the off-flavor compounds. Work this past year demonstrated that dogs were able to smell with 90% accuracy if a fish fillet was off or on-flavor. This research was done in cooperation with Auburn University under a Specific Cooperative Agreement (project number 6420-32000-022-02S). 4b.List other significant accomplishments, if any. A rapid (less than 1 hour) detection assay (loop-mediated isothermal amplification method, LAMP) was developed for the detection of E. ictaluri, the most important disease agent in the catfish industry. LAMP was shown to be rapid, accurate and sensitive (about 10 bacteria/sample) for the detection of E. ictaluri. This assay has the potential to be used for rapid and specific detection of E. ictaluri in catfish from hatcheries and ponds. An indirect fluorescent antibody technique (IFAT) was developed for the detection and identification of Streptococcus iniae. The IFAT was used to positively identify S. iniae from naturally infected tilapia from a fish farm and could identify as few as 10 streptococci in a sample. Due to the highly infectious nature of this disease, a rapid and reliable detection method may help fish farmers make better decisions to more effectively manage this disease in water reuse aquaculture systems. A method for the rapid differentiation of Streptococcus iniae was developed based on the ability of this organism to utilize starch. S. iniae is one of the only streptococcal species to utilize starch and we employed a plate culture technique that could be completed in 24 hours to indicate starch hydrolysis. Flavobacterium columnare was characterized and positively identified from tilapia in Brazil. These new isolates proved to be genetically different from other F. columnare strains described in the literature. Transmission studies demonstrated that Flavobacterium columnare was able to infect fish through the water without the need for fish to fish contact. We were also able to show that the mucus and/or the gill provide the best sites for the detection and/or isolation of F. columnare with traditional methods (i.e., standard plate culture) or molecular biology based assays (PCR). An in vitro (laboratory) method was used to study programmed cell death (apoptosis) of Ichthyophthirius multifillis (a parasite of catfish). We were able to show that antibodies made by the catfish against this pathogen induced the infective stages of the parasite to under go programmed cell death and thus is important in immunity to this pathogen. During the 2004 growing season a total of 1,000 catfish were sampled from experimental ponds at the North Fisheries Experimental Station, Auburn University, Auburn, Alabama. Fish (ten from each of 25 ponds) were sampled in April, June, August, and October for the presence of E. ictaluri and F. columnare by standard methods and polymerase chain reaction. Edwardsiella ictaluri was not detected from the apparently healthy channel, blue or hybrid catfish in these ponds. Flavobacterium columnare was only detected in April by both culture and PCR assay. PCR assay proved more sensitive than standard plate culture techniques in detection of F. columnare from apparently healthy catfish in this study. A secondary objective of this work was to estimate the amount of resources needed to assess E. ictaluri and F. columnare incidence on catfish farms. Our data suggest monitoring E. ictaluri/F. columnare presence in the catfish farm environment by using classical and molecular diagnosis protocols had a cost for reagents and supplies of approximately $7/sample (labor costs excluded). The potential of stress and poor water quality such as low dissolved oxygen to cause increased susceptibility to Streptococcus agalactiae disease has been experimentally evaluated in tilapia. A simple technique using a blood glucose monitor was used to indicate stress in fish (i.e., elevated blood glucose). The research demonstrated a linkage between disease susceptibility and low dissolved oxygen stress. Further, the influence of environmental stressors on fish health (harmful algal blooms and eutrophication) preceeding and following fish kill events has been explored and indicated shifting microbial species between sick and healthy fish. A grant has been submitted to National Oceanic and Atmospheric Administration to explore these environmental interactions in an ecological sense (Cheasapeake Bay) and from a potential human health perspective. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project is the result of splitting the research project entitled, "Integrated Management of Fish Health by Multi-disciplinary Approaches" into 4 new projects based on stakeholders' (fish farmers, aquatic animal health managers, regulatory agencies, etc.) needs. The project entitled, "Aquatic Animal Diagnosis, Pathogenesis and Applied Epidemiology" was certified by the Office of Scientific Quality and Review in November, 2004. The official start date was May, 2005. The project is directly related to the Aquaculture 106 National Program. The potential impact of the research completed under this research project include the development of non-lethal detection methods for fish pathogens and off-flavor compounds, definition of the mechanism of disease (e.g. transmission and virulence) and identification of potential risk factors of disease (epidemiology). The information generated will be used by fish farmers and aquatic animal health managers to improve management decisions regarding fish health. The major accomplishment of this project was the training of dogs to detect off-flavor compounds in water and fish fillets. Off-flavor (an earthy or musty taste) in catfish production costs the industry as much as $50 million annually. Dogs selected from a local animal shelter were trained to sniff water samples for the off-flavor compounds. Work this past year demonstrated that dogs were able to detect these compounds in fish fillets. The dogs were able to smell with 90% accuracy if the fillet was off or on-flavor. This research was done in cooperation with Auburn University under a Specific Cooperative Agreement (project number 6420-32000-022-02S). The potential impact could be a cost saving of tens of millions of dollars due to the potential to predict an off-flavor event and/or only on-flavor fish will be harvested for sale to consumers. 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? The methods for using dogs to detect off-flavor compounds in pond water and fish fillets have been taught to Auburn University extension personnel and are being evaluated for use in the field. The potential constraints on adapting this technology will be processor and farmer acceptance. We also need to determine if the dogs have the ability to retain detection accuracy. Methods for rapid detection of Edwardsiella ictaluri, Flavobacterium columnare and Streptococcus iniae have been published or are in press and are (or will be) available to other scientists working in the area of fish health. The cost of these methods may make them cost prohibitive at this time for use in rapid detection in diagnostic laboratories (i.e. fish already sick). The tests may be useful to detect pathogens prior to disease onset and this may aid farm managers and fish health specialists in making appropriate management decisions. In this case the tests may be cost effective especially because of the rapidity (less than 1 hour) and sensitivity (less than 10 bacteria) of the tests. 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). X.L. Yu, Gan, X., Liang, W.W., Xu, D.H. 2004. Current research on fish parasite Ichthyophthirius multifiliis. Journal of Modern Fisheries Information. 19:14-17. Review Publications Figueiredo, H.C., Klesius, P.H., Arias, C., Evans, J.J., Shoemaker, C.A., Pereira Junior, D.J., Peixoto, M.T. 2005. Isolation and characterization of strains of flavobacterium columnare from brazil. Journal of Fish Diseases. 28(4):199-204. Shoemaker, C.A., Bader, J.A., Nusbaum, K.E., Klesius, P.H. 2004. Detection and distribution of flavobacterium columnare in experimentally infected channel catfish (ictalurus punctatus) using culture and polymerase chain reaction. Journal of Applied Aquaculture. Welker, T.L., Shoemaker, C.A., Arias, C.R., Klesius, P.H. 2005. Transmission and detection of Flavobacterium columnare in channel catfish, Ictalurus punctatus. Diseases of Aquatic Organisms. 63: 129-138. Evans, J.J., Klesius, P.H., Shoemaker, C.A. 2004. Starch hydrolysis testing of multiple isolates for rapid differentiation of streptococcus iniae. Bulletin of the European Association of Fish Pathologists. 24(5)2004:231-239. Xu, D., Klesius, P.H., Shoemaker, C.A. 2005. Cutaneous antibodies from channel catfish immune to ichthyophthirius multifiliis (ich) induced apoptosis of ich theronts. Journal of Fish Diseases 28(4): 213-220.