Thesis(M.S.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

Dissertation (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 51-54).

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.

No description available.

Thesis(M.S.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

Thesis (M.S.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references.

Thesis (M.S.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 62-75).

Visceral toxicosis of catfish (VTC) is a sporadic, often devastating disease in catfish aquaculture, caused by botulinum neurotoxin serotype/E (BoNT/E). The median lethal dose of BoNT/E for channel catfish (Ictalurus punctatus) fingerlings is 13.7 pg/fish. The diagnosis of VTC is suspected if clinical signs and lesions are produced when affected serum is injected into sentinel-catfish and confirmed if this effect is neutralized with BoNT/E antitoxin. However, the assay is complicated in diagnostic cases by small serum samples from affected fish, lack of standardization and availability of small catfish (~10g). Therefore a zebrafish (~0.4g) bioassay for VTC diagnosis was tested and validated. Susceptibility was tested with other serotypes of toxin to help establish zebrafish as a diagnostic model for BoNTs. BoNT/E consist of100kD heavy chain (HC) and 50kD light chain (LC) linked by a disulfide bond. The HC transports the LC into the cytosol of the neuron, where LC (Zn2+-endoprotease) cleaves the SNAP-25 protein thereby blocking the signal transduction at the neuromuscular junction. The HC-based vaccines can induce protective immunity in mammals. To evaluate HC immunogenicity in catfish, rBoNT/E/HC vaccine produced by USAMRIID was tested; this vaccine did not induce a robust antibody response, but western blot analysis demonstrated specific antibody production in 3 of 11 vaccinated fish. We then developed four channel catfish virus (Ictaluridherpesvirus 1, CCV) recombinants expressing synthetic BoNT/E/HC using our established Gateway CCV recombination system to determine if the virus vector could improve the response. Catfish were vaccinated with these recombinants or with a control vector that expressed Escherichia coli beta-galactosidase (CCV-lacZ). No significant protective immunity or BoNT/E antibodies were observed but CCV-lacZ induced a strong antibody response. These results suggest that BoNT/E/HC has low immunogenicity in channel catfish and deviates from the high immunogenicity observed in mammals. To develop a protective vaccine for VTC, it will be necessary to enhance the BoNT/E /HC immunogenicity in channel catfish. Development of recombinant animals which are resistant to BoNT/E was explored as another potential way to prevent VTC. However, the attempts to modify SNAP-25 of zebrafish by genome editing using Transcription Activator-Like Effector Nucleases were unsuccessful.

No description available.