In Southeast Asia, the family Pangasiidae is important for commercial fisheries and aquaculture. Pangasianodon hypophthalmus (striped catfish) is the most economically important species farmed in Vietnam, with a total export value of 1.7 billion USD in 2012. Intensive aquaculture can lead to problems with major outbreaks of disease and Edwardsiella ictaluri and Aeromonas hydrophila represent two important bacterial pathogens in P. hypophthalmus aquaculture. Immunostimulants have proven to be a very useful food additive for the aquaculture industry, since they can be easily fed to fish to enhance their immune response at times of stress and to improve resistance to disease. The immune system of pangasius catfish has not been fully described, despite the recent growth in aquaculture for this species, and little is known about the effects of immunostimulants on disease resistance. Understanding the immune response is very important in order to evaluate the health status of the fish and assist in control of disease (including prevention) so that production levels by the aquaculture industry can be sustained. The aims of this thesis were to develop and standardise methods to elucidate and measure immune responses in P. hypophthalmus and then to use these with relevant disease models (A. hydrophila and E. ictaluri) and immunomodulators (β-glucans from different sources and at different doses) to determine if bacterial diseases can be controlled, and which functional immune responses and immune genes could be correlated with disease resistance. As a variety of different species from family Pangasiidae are economically important for aquaculture, initial work focused on the characterisation of the immunoglobulin IgM molecule in these species, and anti-P. hypophthalmus IgM mAbs were tested to determine if they cross-reacted between different Pangasiidae species (Chapter 2). Although affinity purification of IgM from the different fish species resulted in a purer preparation ammonium sulphate precipitation (14% w/w), the latter proved faster and easier to perform. The heavy (H) and light (L) chains of IgM from P. hypophthalmus were estimated to be 70-72 kDa and 25-26 kDa, respectively, using SDS-PAGE (12.5%). The L chains of IgM in the other Asian fish species examined were similar in molecular weight to P. hypophthalmus, while the H chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. filamentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Sedimentation velocity ultracentrifugation was used to determine the molecular weight of the whole IgM molecule from P. hypophthalmus as an alternative to the more commonly used native gels that are run under non-denaturing conditions, although this technique proved more complex. Anti–P. hypophthalmus IgM monoclonal antibodies (mAbs) cross reacted with all of the Pangasiidae species and were successfully applied in an enzyme-linked immunosorbent assay (ELISA) using mAb 23 to measure serum antibody response of P. hypoophthalmus following experimental infection with A. hydrophila by interperitoneal (I.P.) injection in Chapter 3 and E. ictaluri by immersion in Chapter 4. As P. hypophthalmus is a relatively new aquaculture species, there are few reports evaluating its immune response to pathogens. Thus, functional assays were standardised to evaluate both innate and adaptive immune responses of this species and then these assays used to compare immune response following stimulation with live and killed A. hydrophila. (Chapter3). Four treatment groups of 40 fish per group (53.2 ± 14.8g.) consisting of an untreated control group, a group injected I.P. with adjuvant (Montanide ISA 760 VG) only, a group injected with heat-killed A. hydrophila (1 x109 cfu ml-1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila 2.7 x105 cfu ml-1 were used in the study. Samples were collected 0, 1, 3, 7, 14 and 21 days post injection (d.p.i.) to assess the immune response of fish. The results indicated that challenge with live or/and dead bacteria stimulated the immune response in P. hypophthalmus significantly above control groups with respect to specific antibody titre, lysozyme activity, phagocytosis and plasma peroxidase at 7 or/and 14 d.p.i. Moreover, on 21 d.p.i. total IgM, specific antibody titre and lysozyme activity from both live and dead A. hydrophila challenge groups were significantly different to the control groups. Differential immune responses between live and dead bacterial challenges were also observed as only live A. hydrophila significantly stimulated WBC counts and plasma peroxidase at 3 d.p.i. with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish challenged with dead A. hydrophila showed significantly stimulated respiratory burst activity. Immunostimulants are food additives used by the aquaculture industry to enhance the immune response, and β-glucan is now commonly used for this purpose in aquaculture. In Chapter 4 the effect of the prebiotic β-glucan on the immune response and disease resistance of P. hypophthalmus was evaluated. The fish (60.3 ± 11.7 g.) were fed with a basal diet (control) or diets supplemented with fungal derived β-glucan at concentrations of 0.05 %, 0.1 %, or 0.2 % g/kg for four weeks. Fish fed 0.1 % commercial yeast derived β-glucan were also included as a positive control group. Samples were collected from fish on Days 0, 1, 3, 7, 14, 21 and 28. The results showed that fish fed with the highest two levels of fungal derived β-glucan had enhanced immune responses compared to the control group, with respiratory burst activity on all days examined and lysozyme activity on 7 days post feeding (d.p.f.) being significantly elevated (P<0.05) in the group fed with 0.2 % fungal derived β-glucan, while plasma anti-protease activity on 21 d.p.f., natural antibody titre on 3 d.p.f. and complement activity 7 d.p.f. and 14 d.p.i. were significantly enhanced (P<0.05) in the group fed 0.1 % fungal derived β-glucan. The lowest dose of fungal derived β-glucan (0.05 %) appeared insufficient to effectively stimulate the fish’s immune response. WBC count, respiratory burst, lysozyme activity and complement were useful as an early indication of immunostimulation (1 to 7 days). Four weeks after feeding with the different diets, the fish were experimentally infected with E. ictaluri by immersion using 8 x104 cfu ml-1 for 1 h and mortalities were monitored for 14 days. There was a great deal of variation in the level of mortalities within the four replicate tanks for each dietary group. Although the in vivo challenge results showed no statistical differences between the groups fed on the different diets, the highest mortalities were observed in group fed with the control diet and the lowest mortalities were observed in the groups fed with commercial yeast derived β-glucan and 0.2 % fungal derived β glucan. Immune gene expression following stimulation with β-glucan and challenge with E. ictaluri was investigated in Chapter 5.

A Catfish Management Database (CMD) was developed to analyze data from large commercial catfish farms. The CMD was developed so that data collected by the farm could be used for management of the farm and for identifying some of the risk factors associated with important bacteria diseases. This database was designed to 1) to incorporate production data already being recorded for generating reports for use at weekly managerial meetings focused on feeding rates, feed conversion ratios, mortalities and harvesting events 2) be easily used by a catfish farmer to collect management data in order to analyze production efficiency through a series of farmer defined management reports and 3) provide the farm with easy access to management reports. Additional customized reports can be generated as requested by the farm management. The next objective of this research was to determine pond level risk factors associated with columnaris disease and Enteric Septicemia of Catfish related mortalities. The data from the CMD was used to produce two publications detailing the analysis of the data and production of a univariate and multivariate models of pond level risk factors associated with both diseases. These studies showed some commonly recorded production variables were associated with either columnaris and/or ESC associated mortalities and if monitored could help identify “at risk” ponds prior to disease outbreaks. A study was then conducted to examine the cost associated with mortality on Mississippi commercial catfish farms. The mortalities examined included ponds that had mortalities from columnaris disease, ESC and then any ponds that had mortalities from either. The cost of each disease was determined along with other factors such as pond age, feed conversion ratio and feed cost that influence the profitability of a commercial catfish farm.

The gram-negative bacteria Edwardsiella ictaluri causes significant economic losses in aquacultured fish. Generally considered host-specific to catfish, there are reports of E. ictaluri outbreaks from other aquacultured species, including ornamental fish raised in the southeastern U.S. Thus, a comprehensive phenotypic and genotypic characterization of E. ictaluri isolates from catfish and ornamental aquaculture was warranted. Morphological, biochemical, and protein profiles of catfish and ornamental derived isolates were mostly similar. Plasmid profiles of wild-type isolates were consistent within groups. Analysis of putative anti-microbial resistant isolates from catfish revealed the presence of multi-drug resistant plasmids. Genomic comparisons indicated marked differences among host groups, including unique T4SSs and phage elements among ornamental fish-derived E. ictaluri isolates. An optimal MLSA scheme consisting of eight reference genes was defined, revealing isolates from catfish and ornamental aquaculture form two discrete phyletic lineages. This study advances our understanding of E. ictaluri affecting two important agricultural commodities in the U.S.

Virulent Aeromonas hydrophila (vAh) is a causative agent of motile Aeromonas septicemia (MAS) in catfish. There are limitations in the current therapeutic and preventative strategies against vAh. The pathogenesis of MAS as well as the immune response of catfish to vAh infection are poorly understood. The aim of this study is to: 1) develop a dual live attenuated vaccine against MAS and enteric septicemia of catfish caused by Edwardsiella ictaluri; and 2) evaluate the vAh bacterial load and gene expression patterns in catfish tissues following vAh infection. Previously, six recombinant vAh proteins (outer membrane protein, TonB-dependent receptor, three fimbrial proteins, and an ATPase) were identified to have vaccine efficacy against MAS, and live attenuated E. ictaluri vaccine strain ESC-NDKL1 was identified as an effective vector for expressing vAh antigens. A total of 29 recombinant ESC-NDKL1 strains have been constructed with the integration of one, two, or three genes encoding vAh antigens into the ESC-NDKL1 chromosome. Vaccine efficacy of the constructed strains was evaluated in channel catfish fingerlings. Four recombinant ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL1::atpase::fimMrfG, ESC-NDKL1::fim::fimMrfG, ESC-NDKL1::tdr::fimMrfG, and ESC-NDKL1::fim::ompA) showed significant protection against MAS with relative percent of survival (RPS) values of 55.72%, 60.18%, 61.74%, and 54.81%. Four triple recombinant ESC-NDKL1 strains (ESC-NDKL1::fimMrfG::ompA::fimA, ESC-NDKL1::atpase::fimMrfG::ompA, ESC-NDKL1::fim::fimMrfG::ompA and ESC-NDKL1::atpase::tdr::fim) showed the best protection with RPS values of 77.93%, 63.18%, 67.74%, and 82.35%. To gain a better understanding of vAh pathogenesis, catfish fingerlings were intraperitoneally injected with vAh strain ML09-119. The anterior kidney, liver, and spleen were collected for determination of vAh distribution and expression of thirteen pro-inflammatory, innate, and adaptive immune-related genes using real-time PCR. Results revealed that vAh spread into catfish tissues within 2 hours and peaked at 12 hours post-infection. vAh infection initiated a strong inflammatory response in catfish tissues. Additionally, our research revealed that surviving catfish were able to develop a primary immune response and possibly generation of memory B cells against MAS. Such information will facilitate the development of vaccines and therapeutic drugs for preventing and treating MAS outbreaks in catfish aquaculture.

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.

Vaccination against intracellular pathogens requires generation of pool of memory T cells, which can respond upon infection and mediate immune responses by either killing of infected host cells or induce killing mechanisms in infected cells. T cell-inducing vaccines aim to deliver the antigen to antigen presenting cells (APCs) by presenting on MHC molecules thus bridging innate and adaptive immunity. The intracellular pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is a devastating disease in catfish industry. E. ictaluri can survive in professional phagocytes and use them as an infection source. Two new live attenuated vaccine (LAV) strains, EiDELTAevpB and ESC-NDKL, were developed by our group. However, the role of LAVs in phagocytosis, bacterial killing, and antigen presentation is unexplored. Therefore, further research is necessary to determine immune responses in channel catfish against LAVs. The long-term goal of this project is to identify immunological APC-dependent mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for ESC. The overall goal of this project is to assess the role of three professional APCs, dendritic cells (DCs), macrophages and B cells in the LAV-induced innate and adaptive immune responses in catfish. The central hypothesis is that efficacious LAV strains will enhance phagocytosis and microbial killing, and promote the generation of T cells that regulate and control protective B cell-mediated immunity. The rationale for this research is that more detailed knowledge about phenotype and function of catfish APCs will not only help gain insight into the evolution of vertebrate adaptive immune system but will provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use. In this study, we first identified DC-like cells in immune-related organs of catfish and assessed their expression patterns in lymphoid organs of catfish in E. ictaluri infection. Although WT strain induces the functional inability of DC-like cells in migration and maturation, LAVs strains promote the migration and maturation of DC-like cells for antigen presentation. Two LAVs enhanced the phagocytosis and killing activity in catfish macrophages and B cells. Also, LAVs induce high expression of T cell-related genes without causing inflammation.

Since being first reported in the late 1980ies, the Enterobacterium Edwardsiella ictaluri has rose in prevalence to become one of the two most damaging pathogens affecting the channel catfish industry. Despite this significance of the pathogen, understanding of the development of the disease, especially its route of entry into the host and the earlier stages of the infection, is still incomplete. A series of challenges were conducted using bioluminescent E. ictaluri either by infecting fish through immersion or topical application of the bacteria directly on the intact or abraded epithelium. This showed that abraded fish developed septicemia and died faster than non-abraded ones. Furthermore, results from a co-habitation challenge suggested that the bacterium induced septicemia through the skin instead of becoming water-borne. Finally, a histological technique was developed allowing the determination that the bacteria radiated from the initial skin infection site and penetrated deeper into the tissue as the challenge progressed. These results all suggest that site of abrasion on the skin can act as a route of entrance for the pathogen into the fish, a fact never previously reported. Transposon mutagenesis was also performed to construct a library of 1728 mutants. Screening of this library allowed us to identify 16 genes which inactivation lead to a decrease in the bacterium ability to colonize the epithelium or cause mortality. Sequencing of these genes allowed the identification of RstA/B, a regulator of invasion genes in Salmonella enterica Typhimurium, a putative ribonuclease, similar to a Shigella protein regulating the expression of adhesin and a protein that constitutes the second member of a newly discovered adhesin family. Finally, to investigate the development of the infection, fish were infected by bioluminescent E. ictaluri and sampled at various time points. At each time point, nine organs (gills, muscles, intestine, spleen, liver, stomach, heart, head kidney and trunk kidney) were sampled, and their bioluminescence was measured and half of these organs were homogenized, serial diluted, and plate counts determined. This allowed confirmation of a complex disease pathogenesis during ESC involving a period of intense reproduction in the spleen, anterior and posterior kidneys followed by a sharp increase in the levels of bacteria in the blood.

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.

Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is considered to be the most serious disease occurring in farmed striped catfish (Pangasianodon hypophthalmus) in Vietnam. This disease has had an increasing impact over the last ten years and has been reported to cause 50-90% mortality of stocks during a single outbreak. Data obtained from natural outbreaks of E. ictaluri in striped catfish showed the role of environmental factors in the establishment and progression of this disease. At present, factors affecting the virulence and transmission of E. ictaluri in striped catfish are poorly understood. The central hypothesis of this thesis focuses on the complex picture of the environmental factors and infectivity of E. ictaluri in striped catfish. In this study, 80 isolates of E. ictaluri recovered from natural clinical disease outbreaks occurring in striped catfish farms between 2002 and 2011 located in 4 distinct geographical areas within Vietnam were characterised using a variety of methods. The biochemical profiles showed that E. ictaluri isolates from striped catfish in Vietnam have similar phenotypic characteristics to other E. ictaluri isolates from other infected fish species. These data showed high levels of phenotypic homogeneity between the E. ictaluri isolates investigated. The status of isolates recovered from natural infections over time and from geographically distinct farms was evaluated using pulsed-field gel electrophoresis (PFGE), plasmid profile identification and antibiotic sensitivity tests. The PFGE results showed 6 main groups with a similarity of 82% and the corresponding genotypes of the prevalent isolates illustrated annual differences. Three plasmid groups were identified distributed among the isolates investigated, in which high molecular weight plasmids of approximately 35 and 140 kb were found in two of the groups. Plasmid profiles of the present study did not show any trend of geographical region or year of isolation. The 140 kb plasmid has been considered as a multi-antibiotic resistance plasmid which confers resistance to tetracycline, trimethoprim and sulphonamides. All Vietnamese isolates showed a high level of resistance to Oxolinic acid, Sulfadimethoxine/Ormetoprim (Romet), Oxytetracycline and Amoxicillin. A reproducible bacterial immersion challenge model was developed and the LD60 estimated prior to performing subsequent experimental challenge studies. Fish were exposed to 107 cfu ml-1 of E. ictaluri by immersion for up to 30 seconds, resulting in a cumulative percentage mortality of 63%. Edwardsiella ictaluri was recovered and identified from all the dead and moribund fish during these experiments and affected fish showed similar clinical signs and pathology to those reported from natural E. ictaluri infections. The present study resulted in a successful experimental immersion challenge model for E. ictaluri infection in healthy striped catfish. Cohabitation challenges were also developed and produced 15-40% mortality, typical clinical signs and pathology, and successful recovery of the challenge organism demonstrating horizontal transmission of E. ictaluri in striped catfish. Experimental studies were then conducted to investigate the association between pH or salinity of water and susceptibility to E. ictaluri infection in striped catfish. The first experiments were performed in in vitro conditions in which E. ictaluri isolates were cultured in a variety of pH and salt concentrations. In vivo experiments were then designed where striped catfish were exposed to 107 cfu ml-1 of E. ictaluri for 30 seconds and then held at 4 different water pHs (5.5, 6.5, 7.5 and 8.5) or NaCl concentrations (0, 0.5, 1 and 1.5%). The results of in vitro experiments showed that a pH value between 5.5 to 6.5 and salt concentration between 0-0.5% were optimal for the growth of E. ictaluri. The in vivo experiments demonstrated that the cumulative mortality of striped catfish in water at pH 5 and pH 6 was significantly higher than that of fish maintained in more alkaline water (p<0.05). By contrast, the cumulative mortality of the striped catfish maintained in 0.5% salt concentration was significantly lower than those kept in 0%, 1% and 1.5% salt concentration (p<0.05). Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infections. This study highlighted the use of pH 8.5 and salinity of 0.5% NaCl as a means of decreasing the susceptibility of striped catfish to E. ictaluri. In conclusion, this study used a variety of methods in order to enhance the understanding of the biochemical, biophysical characteristics, plasmid profile and antibiotic resistance as well as the relatedness of E. ictaluri isolates recovered from farmed striped catfish in Vietnam. This study provided two reliable and reproducible bacterial challenge models (immersion and cohabitation) and emphasised the link between pH and salinity with the infectivity and pathogenicity of E. ictaluri in striped catfish.

The catfish industry suffers losses primarily due to enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium columnare, respectively. Understanding the host-pathogen interactions is vital for prevention and eradication of these diseases. Hence, the overall objective of this study was to analyze whole cell proteomes of these two bacteria, and to determine the changes in E. ictaluri protein expression against in vitro iron-restriction and host serum treatment. High-throughput proteomic analysis of these bacteria was conducted using two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry (2-D LC ESI MS/MS) and two-dimentional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-oflight mass spectrometry (2-DE MALDI TOF/TOF). Identified proteins were clustered into functional groups using clusters of orthologous groups, and subcellular locations as well as possible functional relationships were determined. A total of 788 unique E. ictaluri and 621 unique F. columnare proteins were identified, which represented 12 and 28 pathways, respectively. Vertebrate hosts tend to chelate free iron of their body and make the environment hostile for bacteria. Hence, reduced availability of iron may cause significant stress for pathogens and is considered a signal that leads to alteration in virulent gene expression. Similarly, E. ictaluri might use the catfish blood stream effectively for quick systemic invasion. Hence, exposure to catfish serum components might reveal the ability of E. ictaluri to protect against host defense mechanisms. Using two-dimensional difference gel electrophoresis, responses of E. ictaluri due to in vitro iron-restriction and host serum treatment were determined. A total of 50 and 19 proteins were identified to be differentially expressed due to in vitro iron-restriction and catfish serum treatment, respectively. Among the differentially expressed proteins, several putative virulent determinants, immunogenic proteins, chaperones, and housekeeping genes were noted. To initiate functional studies, four differentially expressed E. ictaluri genes (lamB, glyS, malE, and sdhA) were mutated by inrame deletion. Results from this study provided experimental evidence for many predicted proteins. In addition, identification of differentially expressed proteins provided targets for further functional analysis, which could help elucidate pathogenic mechanisms of E. ictaluri.