Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is one of the most important bacterial diseases causing significant economic losses in the US catfish industry. Understanding the virulence mechanisms of E. ictaluri plays a vital role to develop preventives, such as vaccines for the disease. Therefore, further research is necessary to discover the new virulence mechanisms of this pathogen. The long-term goal of our group is to determine the mechanism of E. ictaluri pathogenesis and to develop effective live attenuated vaccines against ESC. The overall goal of this project is to understand the role of Type 6 secretion system (T6SS) in E. ictaluri virulence and determine the safety and efficacy of T6SS mutants in the catfish host. The central hypothesis is that T6SS in E. ictaluri provide an ability to invade the host cells and survive inside of the channel catfish neutrophils and macrophages, and mutation of T6SS genes will cause attenuation of the bacterial virulence. The rationale for the proposed research is that characterization of the T6SS in E. ictaluri will enlighten its role in E. ictaluri virulence, and T6SS genes can be targeted to develop live attenuated vaccines. In this study, we first constructed mutants of individual T6SS genes and a double mutant. The persistence, virulence, and vaccine efficacy of T6SS mutants were determined in the catfish fingerlings and fry infection model. The T6SS mutants Ei?evpC, Ei?evpC?hcp2, Ei?evpD, Ei?evpE, Ei?evpG, Ei?evpJ, and Ei?evpK were significantly attenuated and provided better protection against E. ictaluri 93-146 in channel catfish fingerlings. The role of T6SS mutants in adhesion and invasion of in vitro catfish epithelial indicated that Ei?evpN, Ei?evpO, and Ei?evpP significantly were less adherent and invasive. The survival and replication of T6SS mutants in in vitro catfish peritoneal macrophages cell line showed that T6SS mutants could survive up to 6 hours after phagocyted by catfish macrophages. The survival and resistance of T6SS mutants to stress conditions present in macrophages phagosome showed that hydrogen peroxide could limit the growth of T6SS mutants in BHI and minimal medium. Ei?evpA, Ei?evpH, Ei?evpM, Ei?evpN, and Ei?evpO exhibited a significant growth decrease.

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Catfish aquaculture is the largest cultured food fish industry in the United States, accounting for approximately $375 million in sales annually, with Mississippi leading the industry with 36,200 surface acres of production. However, infectious diseases such as enteric septicemia of catfish (ESC) are decreasing production efficiencies, creating losses of $40-60 million annually. Live-attenuated oral ESC vaccines are effective in preventing ESC infections, but have not been widely adopted by the catfish industry due to the lack of a system to administer the oral vaccine at the scale seen in commercial catfish production systems. An automated system was developed to administer a dosage of 220.5 ml of a live-attenuated ESC vaccine per kg of catfish feed, adapting commercial catfish feeder design to include a screw conveyor for mixing vaccine and feed in a continuous process, pulse-width modulated spray nozzle control for precise vaccine application, and a programmable automation controller to regulate and monitor system performance. Initial performance evaluations demonstrated system operation within the desired design specifications. System feed rates were determined to be a function of the rotational speed (RPM) of the screw conveyor and to be linear across the operational range. Feed rates were observed to decrease by 45% over dry feed when applying liquid vaccine to the feed stream at the 220.5 ml/kg (100 ml/lb) rate, resulting in a feed rate range of 6.80-34.02 kg/min (15-75 lb/min) (95% CI). Uniform pellet-level vaccine distribution is crucial to efficacy as pellet consumption is directly correlated with fish size, with more criticality in smaller fish fed at low rates. Pellet vaccine concentrations at 6.80, 20.41, and 34.02 ml/kg were highly variable and vaccine application at all rates were observed to be statistically different (less) than the target 220.5ml/kg rate (95% CI), pointing to potential issues with vaccine delivery system configuration or inadequacies in sampling methodology. Further evaluation at the pellet level with live-attenuated vaccine to obtain viable cell counts within individual pellets would provide data necessary to address uniformity of coverage questions more fully and to develop operational protocols that maximize system capabilities and vaccine efficacy.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), a devastating disease in the channel catfish industry. Our research group has developed several E. ictaluri live attenuated vaccine (LAV) candidates (EiΔevpB, EiΔevpBΔfur, EiΔevpBΔhfq, EiΔevpBΔfurΔhfq), which were able to stimulate an immune response in vaccinated channel catfish and reduce ESC. However, innate, and adaptive immune responses in the lymphoid tissues of channel catfish to these LAVs are not known well. The overall goal of the project is to determine the role of adaptive and innate immune responses in catfish after vaccination with LAVs. Analysis of innate and adaptive immune-related gene expressions showed that the LAVs induced expression of adaptive immune-related genes in lymphoid tissues with less inflammation compared to wild type control. Also, the LAVs induced the expression of IgM in the sera of catfish.