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.

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.

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is the most economically important disease of farm-raised channel catfish. E. ictaluri is considered a facultative intracellular pathogen like other well-known species in the Enterobacteriaceae, and it is capable of surviving inside channel catfish neutrophils and macrophages. Its ability to survive inside neutrophils and macrophages has made the development of an effective vaccine against ESC particularly challenging. The goal is to develop a safe, efficacious live attenuated ESC vaccine that is practical and economically beneficial to catfish producers. In this study, single and combination of mutations in genes encoding TCA cycle enzyme and C-1 metabolism proteins were constructed using inrame mutagenesis. The virulence, vaccine efficacy, and tissue persistence of the constructed single and combination mutants were determined in channel catfish. The constructed mutants EideltasdhC, Eideltamdh, EideltafrdA, EideltaglyA, EideltasdhCdeltamdh, EideltasdhCdeltafrdA, and EideltasdhCdeltagcvP were significantly attenuated and showed 100% protection against E. ictaluri 93-146 infection in juvenile channel catfish. However, when tested in 15-d old catfish fry, mutant EideltasdhCdeltagcvP and EideltafrdA were found to provide good protection (99% and 60%, respectively) against E. ictaluri 93-146 infection. The tissue persistence study indicated higher tissue concentration in mutants EideltasdhCdeltagcvP and EideltafrdA relative to the tissue concentration in EideltasdhC and EideltasdhCdeltafrdA mutants.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), one of the most important bacterial diseases of farmed channel catfish in the USA. Use of live attenuated vaccines (LAVs) is an effective strategy for combating mortalities in catfish farms. Our research group has developed three live attenuated E. ictaluri strains [EiΔevpB, EiΔgcvPΔsdhCΔfrdA (ESC-NDKL1), and EiΔhemRΔfrdAΔsdhC (triple-hemR)] that provide various levels of protection against ESC. However, the protective mechanisms of these vaccine candidates are mostly unknown. The overall objective of my study was to investigate protective mechanisms of these LAVs. To accomplish this, catfish fry were immersion challenged with EiΔevpB, ESC-NDKL1, and triple-hemR. Additional catfish fry were immersion challenged with Aquavac-ESC and E. ictaluri wild-type (EiWT) as controls. The internalization of antigens through the mucosal surfaces as well as the pathology and molecular immune responses were studied. The investigations showed that EiΔevpB and ESC-NDKL1 were highly safe and efficacious compared to Aquavac-ESC and EiWT. Pathologically, vaccination with EiΔevpB and ESC-NDKL1 decreased the pathological lesions, EiWT replication in catfish, and increased the ability of the immune system to resist and kill EiWT. On the other hand, triple-hemR was not safe causing severe tissue damage similar to EiWT. The gene expression data showed significantly high expression of innate and adaptive immune genes following vaccination and challenge with EiWT. Additionally, EiΔevpB and ESC-NDKL1 induced immune proliferation in pronephros after exposure to EiWT. Taken together, EiΔevpB and ESC-NDKL1 vaccine strains performed better to induce immune responses and reduce damage in the host tissues during EiWT challenge compared to Aquavac-ESC.

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is one of the most important diseases impacting the US catfish industry. Though this disease has been very common, progress has been slow to find an economical and practical treatment method. Our long-term goal is to determine the mechanisms of E. ictaluri virulence in ESC. The overall objective of this study was to identify E. ictaluri genes required for host encounter and serum resistance and to determine their roles in pathogenesis. The central hypothesis is that E. ictaluri must differentially regulate its genes to invade fish and evade host defenses, thus, mutation of these differentially expressed genes (DEG) should cause attenuation of E. ictaluri virulence. To test this hypothesis, we first determined the global gene expression patterns of the wild type (wt) E. ictaluri 93-146 and EiAKMut02 mutant during catfish encounter and serum exposure using microarray analysis. Results indicated that in E. ictaluri wt, 377 and 16 DEGs were identified during host encounter and serum exposure, respectively. In EiAKMut02, 82 and 296 DEGs were identified during host encounter and serum experiment. Through functional analysis using Blast2GO, PSORTb, Host Pathogen Interaction Database (HPIDB), and Microbe Virulence Database (MVirDB), 38 DEGs in 9 KEGG pathways have been identified as potential virulence factors. The KEGG pathways represented were 1) bacterial secretion system including T3SS and T6SS, 2) ABC transporters including cystine transport system, iron complex transport system, d-methionine transport system, arginine transport system, thiamine transport system, and molybdate transport system, 3) protein export, 4) flagellar assembly, 5) two-component system, 6) bacterial chemotaxis, 7) ascorbate and aldarate metabolism, 8) phosphotransferase system, and 9) metabolic pathways. In order to understand their role in the E. ictaluri virulence, selected DEGs were inrame deleted by allelic exchange, and their virulence and efficacy were characterized in channel catfish fingerlings. Our results showed that the virulence of E. ictaluri ssaV and yscR mutants was completely attenuated while their efficacies were moderate in catfish fingerlings. These results support that the T3SS and T6SS, ABC transporters, protein export, and flagella seem to be important in E. ictaluri virulence.

Catfish, the "king" of the U.S. aquaculture, is threatened by a severe, systemic bacterial disease known as enteric septicemia of catfish (ESC). This disease causes high mortality and massive economic losses in cultured channel catfish (Ictalurus punctatus) in the United States. E. ictaluri penetrates catfish intestinal epithelia quickly and establishes a systemic infection rapidly. However, our knowledge on catfish intestine and E. ictaluri interaction is very limited. In Particular, catfish intestinal immune responses and virulence genes needed by E. ictaluri to evade host defenses are not well understood. Hence, our long-term goal is to identify the molecular mechanisms of E. ictaluri-host interactions. The overall objectives of this study were to understand catfish immune responses to E. ictaluri infection and determine essential genes of E. ictaluri during the intestinal invasion. To accomplish the overall objectives of this research, intestinal ligated loops were constructed surgically in live catfish and loops were injected with wild-type E. ictaluri and two live attenuated E. ictaluri vaccine strains developed recently by our research group. We first determined catfish intestinal immune responses against E. ictaluri wild-type and live attenuated vaccine strains. Then, we analyzed the global gene expression patterns of wild-type E. ictaluri and vaccine strains during catfish intestinal invasion using high throughput RNA-Seq technology. Results showed a moderate level of neutrophil and B cell infiltration correlated with significantly lower expression of TNF-α, CD4-1, and CD8-α in the vaccine injected intestinal tissue compared to that of wild-type injected intestinal tissue. Further, RNA-Seq data analysis showed the prominent expression of genes related to bacterial secretion systems, ATP production processes, and multidrug resistance (MDR) efflux pumps in wild-type E. ictaluri. In contrast, the prominently expressed genes in vaccine strains were related to the phosphotransferase system and sugar metabolism processes. All these data suggest that our live attenuated vaccines are capable of triggering effective immune responses in catfish without causing damage to the host.

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.

Proliferative gill disease (PGD) caused by the myxozoan parasite Henneguya ictaluri is one of the most devastating parasitic infections in channel catfish aquaculture. Currently, there is no effective treatment for H. ictaluri and the unpredictable outbreaks can result in 100% mortality. Management strategies have been developed to prevent losses in newly stocked fingerlings by evaluating the PGD status of a pond prior to stocking, which is difficult since resident fish may not show clinical signs even when actinospore levels are lethal to naive fish. Current diagnostic methods are limited to the identification of an active infection and methods of predicting potential outbreaks have several limitations. The PGD status of a pond to be stocked can be determined using sentinel fish exposures which are labor intensive and require a source of parasite free fish. These limitations necessitated the development of more rapid and efficient means of determining actinospore concentrations to determine the risk of losing fish prior to stocking. The development of a quantitative real-time polymerase chain reaction (QPCR) assay provided a more rapid, sensitive and quantitative method of diagnosing active infections and also provides a means to predict potential PD outbreaks and determine the PGD status of a pond prior to stocking. Another approach in the control of this parasite is the identification of a less susceptible culturable species or to identify traits that could be targeted in a selective breeding program. Challenge studies have shown that the closely related blue catfish (Ictalurus furcatus) does not exhibit as severe an inflammatory response to H. ictaluri and mortalities are significantly lower than in channel catfish. Comparisons of PGD severity and H. ictaluri infection in channel catfish, blue catfish and channel x blue catfish backcross hybrids by gross examination, histopathology and the newly developed H. ictaluri real-time PCR (QPCR) assay supported previous research suggesting the life cycle of the parasite can not be completed as efficiently through the blue catfish host. This dissertation describes the development and validation of a QPCR assay to detect H. ictaluri in both fish tissues and environmental samples and the application of this assay in both research and production settings.