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

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC). Although some virulence mechanisms in E. ictaluri have been identified, further research is needed to discover new virulence genes, which could be used to develop safe and efficacious live vaccines. Here, we report production of growth deficient E. ictaluri mutants on complex agar media and identification of genes causing this growth deficiency. The overall goal of this project is to generate growth deficient E. ictaluri mutants and identify genes causing this growth deficiency on complex media. Mutants exhibiting slow growth in complex media may be potential candidates for vaccine development. In this study, 56 unique E. ictaluri genes have been identified. 32 of them showed host protein binding properties while 30 of them were found to be involved in bacterial virulence in other pathogenic bacteria.

This study used rag1-/- mutant zebrafish, which lack functional T and B lymphocytes, to investigate whether innate immune cells from vaccinated mutant zebrafish demonstrate enhanced survival compared to phagocytes from naïve mutant fish. Edwardsiella ictaluri, an economically significant aquatic pathogen and the causative agent of enteric septicemia of catfish (ESC), was used for the trials. Quantification of live bacteria from sampled kidneys was accomplished via colony counts, luminescence readings, and differential DNA extractions using Ethidium Monoazide (EMA) and Propidium Monoazide (PMA) followed by qPCR. There was a general trend of less bacteria in vaccinated mutant fish. Additionally, the mortality in the vaccinated fish was less than the naïve group, suggesting that the vaccinated fish are better able to withstand the bacteria load. Giemsa-stained cytospins showed E. ictaluri exclusively within macrophages from sampled kidneys, suggesting that the macrophages are the critical site of pathogenesis in rag1-/- zebrafish.

Incidence and prevalence of Edwardsiella piscicida has increased in Mississippi farm-raised catfish in recent years. Edwardsiella piscicida affects mostly market-sized catfish during the final stages of the production cycle resulting in significant economic losses. The objectives of this study were to determine the genetic variability of E. piscicida, assess virulence in channel and hybrid catfish, and evaluate the capacity of a live-attenuated E. ictaluri vaccine to protect channel and hybrid catfish against heterologous E. piscicida isolates. This work identified five discrete E. piscicida lineages, along with group specific associations of several virulence related genes. In general, E. piscicida was shown more virulent in hybrids than channel catfish, in line with previous work. Further, a live-attenuated E. ictaluri vaccine was shown to confer cross-protective immunity in channel and hybrid catfish against E. piscicida.

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

Diseases caused by Edwardsiella spp. are responsible for significant losses in wild and cultured fishes around the world. Historically, Edwardsiella tarda has been considered the most phenotypically and genotypically heterogeneous member of the genus. Investigations into intraspecific variability of E. tarda demonstrated isolates previously classified as E. tarda actually represent three genetically distinct yet phenotypically ambiguous groups, leading to the adoption of E. piscicida and E. anguillarum as discrete taxa. Current genomic investigations have demonstrated significant differences between these organisms. To this end, real-time quantitative PCR assays were developed to quickly and accurately detect the pathogens in pond water, fish tissue and broth culture. Additionally, whole genome sequencing was performed for representative isolates of each Edwardsiella spp. Furthermore, forty-seven Edwardsiella isolates, representing all five taxa, from different hosts and a wide temporal and geographic range were analyzed using commercial microbial identification kits, repetitive sequence-mediated polymerase chain reaction, fatty acid methyl ester analysis, antimicrobial resistance profiles, in addition to 16S, gyrB, sodB and plasmid sequencing. This analysis demonstrated key differences in gene sequences and plasmid profiles among these important bacterial pathogens and further supported contemporary taxonomic classifications. Additionally, a real-time multiplex PCR was developed to accurately discriminate between all Edwardsiella spp. affecting fish; namely E. ictaluri, E. piscicida, E. tarda and E. anguillarum. Moreover, recent publications suggest E. piscicida is more commonly associated with disease outbreaks in Mississippi catfish aquaculture than E. tarda or E. anguillarum. To this end, several different challenge models were evaluated. Comparative virulence was assessed, along with histopathological lesions and posterior kidney clearance rates in channel catfish (Ictalurus punctatus). Diagnostic case submissions suggest E. piscicida is more commonly associated with disease outbreaks in blue catfish (Ictalurus furcatus) x channel catfish hybrids compared to channel catfish. This led to investigations into the relative pathogenicity of E. piscicida in hybrid and channel catfish, which demonstrated a significantly lower median lethal dose (LD50) for E. piscicida in hybrid catfish; an important finding given the increased production of hybrid catfish in U.S. farm-raised catfish in the southeastern United States.

Thesis (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 104-128)

Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

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.

Edwardsiella ictaluri is an intracellular Gram-negative pathogen, causing enteric septicemia of catfish (ESC). Universal stress proteins (USP) are important in bacterial virulence, but the role of USPs in E. ictaluri virulence is not explored yet. Our aim was to analyze gene expression of 13 usp (usp01-usp13) and 7 USP-interacting genes (groEL, groES, dnaK, dnaJ, clpB, grpE, and ppGpp) under low pH, H2O2, catfish serum, and in vivo stress conditions, construct USP mutants, and determine mutants’ role in E. ictaluri virulence. We found that usp05, usp07 and usp13 genes were highly expressed under all stress conditions, while groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Among the 10 E. ictaluri USP mutants, Eiusp05-07-08-09-10, and 13 were significantly attenuated in catfish and highly protective against wild type E. ictaluri infections in catfish. Eiusp05-07-08-09, and 13 were sensitive to oxidative stress, and all mutants were sensitive to pH exposure.