2007 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research project is to conduct aquaculture research addressing fish health problems limiting the production of channel catfish, Ictalurus punctatus. 1b.Approach (from AD-416) All research conducted under this agreement will address practical, production oriented fish health or disease problems facing the farm-raised catfish industry in the southeastern United States and will address components of the USDA-ARS action plan for National Program 106-Aquaculture. Research projects will be conducted using pond and laboratory facilities at the Delta Research and Extension Center (National Warmwater Aquaculture Center, Stoneville, MS). These facilities are under the direction of the Mississippi Agricultural and Forestry Experiment Station (MAFES) and Mississippi State University College of Veterinary Medicine (CVM). Research will address problems in the following broad subject areas: fish health management, fish diagnostics, epidemiology, and risk assessment modeling. On-farm demonstration projects, scientific articles, technical bulletins and educational workshops will be used to disseminate information to stakeholders. 3.Progress Report This report documents research conducted under a Specific Cooperative Agreement between MS Agricultural & Forestry Experiment Station (MAFES) and ARS. Additional details for this subordinate project may be found in the in-house project 6402-31630-001-00D, “Improving Production Efficiency of Pond-Raised Channel Catfish.” Objective 1: Develop diagnostic tools to detect and monitor diseases in commercially raised channel catfish and determine virulence factors associated with those diseases. Hypothesis 1: A real-time PCR assay can be developed to study the epidemiology of proliferative gill disease. Proliferative Gill Disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is one of the most devastating parasitic infections affecting commercial channel catfish (Ictalurus punctatus) aquaculture. To evaluate the pathogenesis of infection and predict potential losses associated with this disease a real-time polymerase chain reaction (QPCR) assay was developed using published H. ictaluri specific end point PCR primers and protocols. The sensitivity threshold of the assay was less than 1 H. ictaluri actinospore and there was no subsequent amplification of the genomic DNA of several closely related myxozoans commonly found in channel catfish ponds. Disease monitoring studies are being conducted to evaluate the prevalence and severity of PGD in commercial catfish production ponds. Further application of this assay will involve establishing a critical H. ictaluri actinospore concentration to identify the threshold at which ponds are safe to stock and evaluating potential chemotherapeutic and biological treatments Hypothesis 2: A real-time immuno-PCR assay can be developed to study the epidemiology of enteric septicemia of catfish. An “immuno-capture” system to extract E. ictaluri or F. columnare cells from pond water has been developed and used successfully in laboratory settings, but has been unsuccessful in field applications. A water filtration method using the real time PCR assay has been developed in substitution of the “immuno-capture” method. The procedure is being optimized for field applications. Hypothesis 3: Early stages in the pathogenesis of enteric septicemia can be elucidated by examining differential protein expression by Edwardsiella ictaluri exposed to channel catfish intestinal mucus. We have found that in pond water and when exposed to catfish tissues and mucus E. ictaluri immediately adheres to larger particles. Isolating the bacteria from the host tissues and mucus without changing the protein expression of the bacteria has proven difficult. An “immuno-capture” system to extract E. ictaluri cells from pond water and catfish tissues has been developed, and is being optimized to isolate the bacteria with little or no host materials. Once a method of isolating the bacteria from the catfish tissue and mucus components has been optimized the characterization of the bacterial proteins expressed in vivo and in pond water will proceed. Project was monitored by regular discussions and interactions between the MAFES staff and the CGRU. This project continues work of 6402-31630-001-02S that terminated in May 2007.