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? 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 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 managers 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 (PCR) and detection 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 by year the currently approved milestones (indicators of research progress) 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 (2005-2009); . 2)field testing of dogs for the detection of off-flavor compounds (2005-2006);. 3)development of in vitro models to assess monogenetic trematode infections (2006-2008);. 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 (2005-2008);. 5)The milestones for the epidemiology studies include the development of protocols and methods to investigate F. columnare/E. ictaluri presence in channel catfish (2006-2009); and. 6)conduct studies to assess the relationship between harmful algae, stress and bacterial infection in aquatic animals (2005-2009). 4a.List the single most significant research accomplishment during FY 2006. The deceptive marketing of imported basa, Pangasius boucourti, fillets as catfish has resulted in serious economic losses to the farmed catfish industry in the US. The fillets appear similar following processing and a rapid method to differentiate uncooked, cooked and/or marinated channel catfish fillets from basa and other filleted fish was needed. A monoclonal antibody specific for a channel catfish fillet protein was produced and characterized. The monoclonal antibody was used to develop an indirect enzyme linked immunosorbent assay (ELISA) specific for fish of the species Ictalurus (i.e., channel catfish, blue catfish and hybrid channel X blue catfish). The assay correctly identified raw or cooked channel catfish fillets in less than 3 hours from other species (basa, striped bass, sea bass, red snapper, tilapia, flounder) tested in a single blind study. 4b.List other significant research accomplishment(s), if any. A rapid (less than 1 hour) detection assay (loop-mediated isothermal amplification method (LAMP)) was developed for the detection of F. columnare, the most second most important disease agent in the catfish industry. LAMP was shown to be rapid, accurate and sensitive for the detection of F. columnare. This assay has the potential to be used for rapid and specific detection of F. columnare in catfish from hatcheries and ponds. An immunofluorescent technique was developed that can be used to simultaneously determine the presence of Flavobacterium columnare and Edwardsiella ictaluri in channel catfish. This technique uses monoclonal and polyclonal antibodies to make rapid diagnosis of these diseases possible. This method will facilitate epidemiology studies as it may allow non-lethal sampling using a swab of the gills or skin mucus. Detection of off flavor compounds using trained dogs. Three dogs were tested and proven to be a sensitive and accurate method of determining off flavor in catfish fillets (about 90%). Two additional dogs were transferred to the Alabama Fish Farming Center where they were made available to local producers for pond sample testing. Producer response to the method was met with interest, but not acceptance as a routine procedure. New technology using small rapid chromatographic instruments known as "electronic noses" may provide a more acceptable rapid method for the detection of off flavor volatiles in pond water and fillets. A technique utilizing whole cell fatty acids in the cell wall of Flavobacterium columnare was developed to identify pure cultures of F. columnare using a gas chromatography system. This method can positively identify 24-48 h cultures of F. columnare in about 10 minutes following extraction of the fatty acids. Flavobacterium columnare presents a unique profile that allows identification to the species level. The old method (i.e., standard biochemical tests) required about 7 days to complete a positive identification. Molecular epidemiology of Flavobacterium columnare has been explored. New F. columnare isolates have been incorporated into the existing database during FY 2006. After species confirmation by fatty acid methyl ester analysis (FAME) and specific polymerase chain reaction (PCR), F. columnare isolates were fingerprinted using amplified fragment length polymorphism (AFLP) and intergenic spacer region (ISR) sequence. F. columnare cultures isolated at the Fish Diagnosis Laboratory (Auburn University) that presented unique morphological or phenotypical traits were subjected to polyphasic characterization (approximately 10 new clinical strains were incorporated to the collection). In order to expand our current database, around 100 new F. columnare strains were collected from wild fish during FY 2006. Since most of our archived isolates came from farm raised fish, the addition of environmental isolates from natural populations is crucial to fully understand F. columnare intraspecies diversity. These new isolates have been AFLP-typed and ascribed to different genomovars based on 16S rDNA analysis. In FY 2006, a new fingerprinting method was developed. This new technique is based on single strand conformation polymorphisms (SSCP). We are testing this method by using the ISR sequence as target as well as the 16S rDNA gene. We expect SSCP analysis will provide us with a similar resolution level as ISR sequencing. ISR-SSCP analysis is less expensive than ISR sequencing and results can be obtained in 8 h. New and archived F. columnare isolates were typed by ISR-SSCP as well as by 16S-SSCP. Preliminary data suggest that ISC-SSCP will be a cost-effective method to rapidly fingerprint F. columnare isolates. The microbial communities present in catfish ponds have been characterized by using molecular methods such as 16S gene library construction and Automated Ribosomal Internal Spacer Analysis (ARISA). We were able to show which bacterial species are present in this aquatic environment and how microbial populations differed from pond to pond at any given time. A broad diversity in bacterial species composition was found by 16S rDNA analysis. Gammaproteobacteria was the most represented class in all ponds, followed by alphaproteobacteria and Gram positive high G+C content bacteria. Uniqueness of bacterial communities from each individual pond was confirmed by ARISA. Two fish pathogens, F. columnare and E. tarda were detected in eight ponds in absence of epizootics. Streptococcus spp. are widely distributed and are potential threats to fish and mammals worldwide. We have developed and published improved rapid diagnostic methodologies and technology to detect and identify fish pathogenic bacteria S. iniae and Vibronaceae. Furthermore, we have characterized Streptococcus and Lactococcus from a marine mammal suggesting that host shifts may be plausible. Gyrodactylus is a parasite that can cause severe loss of cultured young catfish and tilapia directly (i.e., mechanical injury of fish) or indirectly (i.e., aid in bacterial infection). To better understand the invasion mechanism and control of the parasite, methods for culturing and maintaining Gyrodactylus under laboratory conditions and methods for harvesting the parasite were developed. These methods were used to. 1)evaluate mortality and infection of Nile tilapia parasitized with Gyrodactylus and exposed to Streptococcus iniae and. 2)determine if Gyrodactylus transmit S. iniae from infected to un-infected tilapia. Techniques have been perfected to allow for survival of the parasite for about 3-4 day in vitro. Previous results suggested survival for only 24 h without a fish host. The influence of environmental stressors on fish health has been explored and has indicated that unlike exposure to sublethal levels of dissolved oxygen, sublethal levels of unionized ammonia alone does not increase disease susceptibility to S. agalactiae. Other experimental work has elucidated that the non traditional host, catfish, is susceptible to both streptococcal species, S. iniae and S. agalactiae and the role of concurrent natural parasitic Trichodina spp. infestation may increase this disease susceptibility. These studies are aiding the understanding of the probability of disease epizootics under varying stressor conditions and in alternative hosts. Transmission routes and pathogenesis have been explored in both E. tarda and S. agalactiae infectivity studies in channel catfish and tilapia, respectively. Paternal transmission of Streptococcus agalactiae and development of skeletal deformities in Nile tilapia, Oreochromis niloticus was documented. 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 potential impact of the research completed under this project includes 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. Significant progress has been made in the development and improvement of methods for detection and identification of bacterial pathogens of fish and for the positive identification of channel catfish fillets. These methods include immunological (i.e., indirect fluorescent antibody tests for E. ictaluri, F. columnare and S. iniae or enzyme linked immunosorbent tests to identify catfish Ictalurus sp. fillets), phenotypic methods (i.e., starch testing of S. iniae; whole cell fatty acid analysis of F. columnare) and DNA based methods (i.e., PCR; LAMP; AFLP; SCCP). The methods have been published and requests for reprints have been received. Fish health diagnostic laboratories (Auburn University, University of Arkansas at Pine Bluff) and researches involved in epidemiological studies have incorporated some of the developed techniques and methods into routine use. An additional accomplishment of this project was to assess the prevalence of F. columnare in experimental catfish ponds using different diagnostic methods. The study showed a low incidence of F. columnare in asymptomatic catfish throughout the study by culture and PCR-based methods. However, a higher indirect presence of this bacterium was assessed by ELISA. Twenty-three percent of total fish analyzed were seropositive for F. columnare. Outbreaks due to columnaris diseases were scarce during the sampling period indicating the mere presence (either direct or indirect) of this pathogen in the catfish farm environment does not suggest a higher disease risk. 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 transferred to the Alabama Fish Farming Center in Greensboro, AL, and are being evaluated for use in the field. The potential constraints on adapting this technology will be processor and farmer acceptance. Methods for rapid detection of Edwardsiella ictaluri, Flavobacterium columnare and Streptococcus iniae have been published and are 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. 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. Review Publications Shoemaker, C.A., Arias, C.R., Klesius, P.H., Welker, T.L. 2005. Technique for identifying Flavobacterium columnare using whole cell fatty acid profiles. Journal of Aquatic Animal Health 17: 267-274. Pasnik, D.J., Evans, J.J., Klesius, P.H. 2005. Nile tilapia, oreochromis niloticus, blood agar and the culture of fish bacterial pathogens. Bulletin of the European Association of Fish Pathologists. 25(5):218-224. McNulty, S.T., Klesius, P.H. 2005. Development of an indirect enzyme-linked immunoabsorbent assay using a monoclonal antibody to identify channel catfish, Ictalurus punctatus (Rafinesque), fillets. Aquaculture Research 36, 1279-1284. Delaney, M.A., Klesius, P.H., Shelby, R.A. Cortisol responses of Nile tilapia (Oreochromis niloticus (L.)to rapid temperature changes. Journal of Applied Aquaculture. 16: (3/4) 95-104. Yeh, H., Shoemaker, C.A., Klesius, P.H. 2006. Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method. Journal of Applied Microbiology, 100 (2006) 919-925. Evans, J.J., Pasnik, D.J., Brill, G.C., Klesius, P.H. 2006. Un-ionized ammonia exposure in Nile Tilapia: Toxicity, Stress Response, and Susceptibility to Streptococcus agalactiae. North American Journal of Aquaculture. 68(1):23-33. Shelby, R.A., Myers, L.J., Schrader, K.K., Klesius, P.H. 2005. Detection of off-flavour in channel catfish (Ictalurus punctatus Rafinesque) fillets by trained dogs. Aquaculture Research 2005, 37, 299-301. Bader, J.A., Moore, S.A., Nusbaum, K.E. 2006. The effect of cutaneous injury on a reproducible immersion challenge model for Flavobacterium columnare infection in channel catfish (Ictalurus punctatus). Aquaculture 253: 1-9