2008 Annual Report 1a.Objectives (from AD-416) Objective. 1)Develop and apply rapid non-lethal methods to detect, diagnose and evaluate effects of pathogens and toxins in aquatic animals. Objective. 2)Develop in-vivo and in-vitro models for addressing the pathogenesis (mechanism of disease) of bacteria, toxins, parasites and viruses in aquatic animals. Objective 3a) Determine and assess the risk factors associated with disease through epidemiologic studies; b) Determine the disease prevalence, incidence, sources and origin of economically important aquatic animal pathogens, especially, Edwardsiella ictaluri, E. tarda, Flavobacterium columnare, Streptococcus iniae, S. agalactiae and Ichthyophthirius multifiliis. 1b.Approach (from AD-416) The 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 specific objective of the project include: . 1)develop and apply rapid non-lethal methods to detect, diagnose and evaluate the effects of pathogens, off-flavor compounds and toxins in aquatic animals;. 2)develop in vivo and in vitro models for addressing the mechanisms of disease for bacteria, parasites and toxins in aquatic animals and. 3)determine risk factors and disease prevalence, incidence, sources and origin of economically important aquatic animal pathogens, especially, Edwardsiella ictaluri, E. tarda, Flavobacterium columnare, Streptococcus iniae, S. agalactiae, Ichthyophthirius multifiliis and Henneguya icatluri (cause of PGD). 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. The approach used for off-flavor compound detection will be canine olfactory detection. Basic studies on the mechanism of disease will be set up to address both horizontal and vertical transmission of the above disease agents. Studies will also be designed to address the effect of toxic compounds on fish in vivo (water borne exposure) and in vitro (i.e. on cell lines or primary cell cultures). The final objective will be completed using a multidisciplinary approach including microbiology, parasitology, immunology, histology and nutrition to answer questions on epidemiology. Meeting the objectives of this multifaceted project will provide information that can be used by fish farmers and aquatic health managers to help reduce the economic impact of health problems in aquatic animal production. 3.Progress Report Validation of a standard Polymerase Chain Reaction (PCR) for detection of Edwardsiella ictaluri. Hundreds of unbiased samples may be required to accurately estimate prevalence or incidence of infection for a pathogen endemic on a catfish farm. Freezing fish on dry ice and storage at -80 degrees centigrade for assay at a later time may be a viable method. A study is in progress to estimate the level of agreement between results for fresh and frozen catfish fingerlings infected in the laboratory with E. ictaluri and assayed by bacterial culture and standard PCR. Diagnostic sensitivity for the PCR assay before and after freezing will be estimated. This information will be used in the design of epidemiological studies to calculate sample sizes required for estimating prevalence and incidence of infection. Egg-associated transmission of Flavobacterium columnare. Infection of fish at the egg stage may result in death at an early or later age. Subclinical infection may also be a consequence and thus result in the introduction of carrier fish into the production cycle. The possibility that egg-associated transmission of F. columnare can occur has not been published. Studies are in progress to sample catfish egg masses (using bacteriological and molecular biological techniques) and assay the eggs for F. columnare. Management strategies for control of endemic infection on the fish farm will result. Epidemiology of disease agents that infect cultured fish. Vaccination should control endemic infection in cultured fish populations. In March, 2007, the commercial columnaris vaccine that was developed at this laboratory was used to vaccinate catfish fry at an Alabama farm. However, due to the negative impact of the drought and economic factors on the study conditions, the cooperation was terminated in the first half of FY 2008. The columnaris vaccine was also used to vaccinate half of the brook and brown trout fry at a North Carolina farm in March, 2007, and additional fry were vaccinated in January, 2008. The difference in mortality due to columnaris disease will be compared between vaccinated and unvaccinated fish. Molecular epidemiology of Streptococcus agalactiae. Despite being known mainly as a mammalian disease agent, S. agalactiae has become recognized as an emerging pathogen of wild and cultured fish. This study examined the genetic relatedness of fish, dolphin, human and cattle S. agalactiae isolates from different geographic regions using serological and multilocus sequence typing. Results suggest that fish isolates are divided into six genetic types. Conduct studies on harmful algae’s relationship to fish health. The relationship between frequency of skin ulceration and total plasma immunoglobulin levels (an indicator of health status) in Atlantic menhaden from Delaware and Maryland inland bays was studied. Data suggest that skin ulceration is correlated with low total immunoglobulin levels and thus these fish are immunosuppressed. NP 106, Component: 4, Problem Statment: b, c, d, e, f, g. 4.Accomplishments 1. Genetic diversity and relationship to virulence found in Flavobacterium columnare. Flavobacterium columnare is pathogenic to many species of freshwater fish throughout the world, and the United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted. F. columnare isolates recovered from diseased channel catfish belong to either genomovar (genetic type) I or II. Genomovar II isolates were shown to be more virulent by immersion (natural route of exposure) in channel catfish fry and fingerlings than genomovar I isolates. The ability of the genetically characterized F. columnare to adhere to the fish was also correlated to increased mortality in challenged catfish. Interestingly, environmental isolates obtained from apparently healthy wild channel catfish (Mobile River basin) were virulent in laboratory studies. Studies suggest that F. columnare from the normal aquatic bacterial community is probably the source of pathogenic F. columnare. This work was done in cooperation with Auburn University under Specific Cooperative Agreement. NP 106, Component: 4, Problem Statment: b, c, d, e, f, g. 2. Effect of tricaine methanesulfonate on survival of fish parasite Ichthyophthirius. Theronts and trophonts of Ich are commonly used to conduct efficacy/toxicity tests for screening chemotherapeutants against Ich, in immunization studies as antigens and for assessing protective immunity. When fish infested with Ich are anesthetized with tricaine to collect the parasite, trophonts come into contact with the anesthetic compound. No information was available on the effect of tricaine on Ich trophonts and tomonts. A study was designed to determine the effect of varying concentrations of both buffered and unbuffered tricaine on the survival and reproduction of Ich tomonts. Results of the study demonstrated that use of tricaine to anesthetize fish had no adverse effect on survival and reproduction of Ich when used as concentration recommended for fish anesthetization. The use of tricaine also makes fish easier to handle, thus decreasing fish stress during the experimental process. NP 106, Component: 4, Problem Statment: d, g. 3. Molecular typing of Streptococcus agalactiae isolates from fish accomplished. The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using two fingerprinting techniques (single-stranded conformation polymorphism analysis of the intergenic spacer region (SSCP-ISR) and amplified fragment length polymorphism (AFLP) analysis. Results demonstrated considerable genetic variability was seen in the S. agalactiae cultures tested. AFLP generated 10 genetic types within the S. agalactiae species whereas, SSCP-ISR generated only 5 genetic groups. This work was done in cooperation with Auburn University under Specific Cooperative Agreement. NP 106, Component: 4, Problem Statment: d, e, f, g. 4. Effect of aquaculture production noise on growth and disease resistance in fish examined. Intensive aquaculture production systems often use equipment (such as pumps, air blowers, filtration systems) that increase noise levels in the fish culture tanks. This increased noise may negatively affect the auditory system, increase stress and ultimately reduce fish performance. Rainbow trout were subjected to noise levels commonly seen in recirculating aquaculture systems. The results of the study suggest that rainbow trout hearing sensitivity, growth, survival, stress and disease susceptibility were not impacted by noise levels associated with recirculating aquaculture systems. This was a collaborative study between the Freshwater Institute and the University of Maryland and ARS. NP 106, Component: 4, Problem Statment: d, g. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 3 Review Publications Shoemaker, C.A., Olivares-Fuster, O., Arias, C.R., Klesius, P.H. 2008. Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus puncatus). Veterinary Microbiology 127: 353-359. Olivares-Fuster, O., Baker, J.L., Terhune, J.S., Shoemaker, C.A., Klesius, P.H., Arias, C.R. 2007. Host-specific association between Flavobacterium columnare genomovars and fish species. Systematic and Applied Microbiology. 30: 624-633. Shoemaker, C.A., Klesius, P.H., Lim, C.E. 2007. Feed deprivation increases the susceptiblity of channel catfish to columnaris disease. Schering Plough Animal Health Aqua Focus 65: 1-4. Davidson, J., Frankel, A., Ellison, W., Summerfelt, S., Popper, A.N., Mazik, P., Bebak, J.A. 2007. Minimizing noise in fiberglass aquaculture tanks: Noise reduction potential of various retrofits. Aquacultural Engineering. 37: pgs. 125-131. Wysocki, L.E., Davidson, J., Smith, M.E., Frankel, A., Ellison, W., Mazik, P.M., Popper, A.N., Bebak, J.A. 2007. Effects of aquaculture noise on hearing, growth, and disease resistance of rainbow trout Oncorhynchus mykiss. Aquaculture. 272:687-697. Clingerman, J., Bebak, J.A., Mazik, P.M., Summerfelt, S.T. 2007. Use of avoidance response by rainbow trout to carbon dioxide for fish self-transfer between tanks. Aquacultural Engineering. 37(3): 234-251. Olivares-Fuster, O., Klesius, P.H., Evans, J.J., Arias, C.R. 2008. Molecular typing of Streptococcus agalactiae isolates from fish. Journal of Fish Diseases. 31(4):277-283. Evans, J.J., Pasnik, D.J., Horley, P.J., Kraeer, K., Klesius, P.H. 2008. Aggression and mortality among Nile tilapia (Oreochromis niloticus) maintained in the laboratory at different densities. Research Journal of Animal Sciences. 2(2):57-64.