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(ies). Several areas of inadequate research are: rapid identification of pathogens, epidemiology of infectious diseases, pathogenesis (i.e. mechanism of disease) of aquatic animal pathogens, and vaccine effectiveness in field studies. The overall objective for this project was to increase our knowledge on bacterial catfish pathogens and to provide fish health specialist with better tools for catfish diseases diagnosis and better management practices. 2.List by year the currently approved milestones (indicators of research progress) The milestones to be addressed include: 1) Evaluation of enteric septicemia of catfish (ESC) and columnaris vaccines efficacy in the field. 2) Development of new detection methods for catfish pathogens. 3) Polyphasic characterization of catfish pathogens: construction of epidemiological databases. 4) Assessment of proliferative gill disease (PGD) in commercial catfish ponds and impact to production. 5) Understanding columnaris pathogenesis: identification and characterization of virulence genes. 6) Identification and characterization of virulence genes in the catfish parasite Ichthyophthirius multifiliis. 7) Modeling long term approaches to control enteric septicemia of catfish. 4a.List the single most significant research accomplishment during FY 2006. The single most significant research accomplishment in the last year shows that catfish fry production (survival) can be increased by 43% using a secondary nursery phase prior to being stocked into fingerling production ponds. From this same experiment, data verifies that incorporating the use of a live, attenuated vaccine produced by vaccine manufacture and patented by the Aquatic Animal Health Research Unit USDA/ARS can in fact increase survival up to 15% in ponds. 4b.List other significant research accomplishment(s), if any. An additional significant achievement of 06 is the successful construction of the I. multifiliis normalized library. We have collected biological samples from the three stages of life cycle of the parasite Ichthyophthirius multifiliis, tomont, trophont, and theront. The total RNA and mRNA was isolated and pufified from these samples for the construction of cDNA libraries. A normalized cDNA library has been successfully constructed that subtracted the highly expressed genes while enhancing representation of rarely expressed genes. After construction of the normalized cDNA library, it was characterized as to the insert size, normalization successes, and insertless background clones. After the characterization of the library, analysis of expressed sequence tags (ESTs) is being conducted. With well normalized cDNA library, we expect to sequence a total of 10,000 EST clones to obtain a high number of unique genes. The sequencing of ESTs are under way. The first step for the transcriptome analysis is to generate ESTs representing a major fraction of the true transcriptome. We plan to sequence 10,000 clones. The organism has perhaps no more than 10,000 genes and hope through EST analyses, we can capture more than 50% of all its genes. Another accomplishment of this project was to asses the prevalence of F. columnare in experimental catfish ponds using different diagnosis 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 increase a higher disease risk. the construction of the F. columnare subtractive hybridization library. 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 research accomplishment that catfish fry survival can be improved from 47% to 67% via the use of a secondary nursery phase prior to being stocked into fingerling ponds. Field efficacy for ESC vaccine, a live attenuated vaccine against Edwardsiella ictaluri, showed that catfish fry survival can be improved by up 15%. Fish vaccinated in the fry stage also have better survival in the food fish stage grown under a single batch cropping system. This accomplishment may have impact on the application of vaccine against E. ictaluri on farms using secondary nurseries to improve fry survival. This data verifies that ESC modified live vaccine can in fact increase survival up to 15% in ponds. The field evaluation of ESC vaccine will help substantially cut down on ESC disease losses. 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 molecular biology and vaccine research accomplishments have been disseminated to other scientists and industry through scientific presentations and publications. The immediate benefits of the research will be realized by other scientists, nationally and internationally. These studies complement the molecular biological and vaccine studies being conducted at ARS, Aquatic Animal Health Research Unit at Auburn, AL, and health research programs at the Catfish Genetics Unit in Stoneville, MS. 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). Schneider, J.E., Jr., J.S. Terhune, and J.M. Grizzle. BlebFormation and XTT Reduction in Channel Catfish Hepatocyte Primary CultureExposed to Microcystin-LR. 2005. The 45th Annual Meeting of the Aquatic Plant Management Society, San Antonio, Texas. Schneider, J.E., Jr., B.H. Beck, J.S. Terhune, and J.M. Grizzle. 2005. Cytotoxicity of Microcystin-LR to Primary Cultures of Channel CatfishHepatocytes and to the Channel Catfish Ovary Cell Line. First International Symposium on Cyanobacterial Harmful Algal Blooms, ResearchTriangle Park, North Carolina. Carrias, A., J. Terhune C. Sayles, and J. Chappell. 2006. Evaluation of an ESC vaccine and nursery phase in the production of channel catfish fingerlings. Aquaculture America: Annual Meeting of the U.S. Chapter of the World Aquaculture Society. Las Vegas, NV. Arias, C., C. Shoemaker, W. Daniels, R. Shelby, T. Welker, J. Terhune, P. Klesius. 2006. Prevalence of Edwardsiella ictaluri and Flavobacterium columnare in channel, blue and hybrid catfish raised in experimental ponds. Aqua 2006: Annual Meeting of the World Aquaculture Society. Florence, Italy. McNeely, J. P., and J. S. Terhune. 2006. Evaluation of an AQUAVAC-ESC® booster vaccine on production of food-size channel catfish Ictalurus punctatus in earthen ponds. Aqua 2006: Annual Meeting of the World Aquaculture Society. Florence, Italy.