β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Vibrio salmonicida causes cold-water vibriosis in salmon populations around the world and causes financial damage to fisheries designed to farm these salmon. Very little is known about the physiology of how V. salmonicida causes disease and measures to contain vibriosis are restricted to either vaccinating individual fish against disease or administering antibiotics when an outbreak is detected. These procedures are costly and increase the risk for selection of antibiotic-resistant V. salmonicida strains. A recent reoccurrence of outbreaks in Norwegian fisheries provided incentive to better understand the virulence mechanisms of V. salmonicida. In this thesis, a proteomic approach was used to identify proteins that were differentially expressed when cells were grown in vitro under simulated virulence conditions (i.e. 5˚C and in the presence of exogenously supplied autoinducer 3-oxo-hexanoyl-homoserine lactone). Some examples of proteins with significantly altered expression that stood out at as homologs of potential virulence factors were: an exported serine protease DegQ, a multi-drug transporter HlyD, and an outer membrane protein OmpU. The proteomic approach allowed us to identify large numbers of proteins that are expressed by V. salmonicida, facilitating hypothesis-driven research in order to support possible roles for some of these proteins in virulence

High pressure processing (HPP) and gamma irradiation were applied to inactivate <i>Vibrio vulnificus</i> (MO624) and <i>Vibrio parahaemolyticus</i> (O3:K6 TX2103) in pure culture and in inoculated live oysters. <i>Vibrio</i> pure culture and inoculated oysters were exposed to pressures of 207 MPa (30 kpsi) to 552 MPa (80 kpsi) for 0 min to maximum of 20 min. More than 5.4 log reductions of <i>V. vulnificus</i> occurred at 345 MPa for 0 min in oysters; 345 MPa for 2 min can achieve 4 log reductions on <i>V. parahaemolyticus</i>. Dosage of 1 kGy gamma-irradiation was proved to be effective in producing <i>Vibrio</i> free oysters with comparable organoleptic quality to raw oysters. Thermal conductivity of shucked oysters was measured to be 0.58 to 0.68 W/m°C, as temperature increased from 0 to 50 °C, using a line heat source probe. The specific heat was measured by differential scanning calorimeter methods. It increased from 3.80 to 4.05 kJ/kg °C, when temperature rose from 10 to 50 °C. The thermal diffusivity was calculated employing the data of thermal conductivity, specific heat and density of shucked oysters. The results showed that, under the tested temperature range, thermal properties did not change significantly with temperature. The dielectric constant and loss factor of oysters were determined by an open-ended coaxial line probe connected to a network analyzer at frequency of 30 MHz to 3000 MHz from 1 to 55 °C. The penetration depth of dielectric heating was calculated to be 1.1 cm with the dielectric constant of 55 and loss factor of 14. A two-dimensional mathematical model was established to simulate the heat transfer of microwave heating using a fish gel. Finite difference method was utilized to solve partial differential heat transfer equations. The model was able to predict the temperature distribution in heated fish gel with an accuracy of ± 8°C. Applying the developed mathematical model, the lethality of <i>Vibrio</i> spp., artificially inoculated in live oysters, was estimated collectively by integrating the individual localized lethality of designated heating units. The predicted lethality was compared with microwave enumeration data on Vibrios in oysters. The observed maximum log reductions by microbial enumeration were 4.4 and 3.4 for <i>V. vulnificus</i> and <i>V. parahaemolyticus</i>, respectively. The lethality calculated by integrating temperature profiles was acceptable. The discrepancy between the estimated lethality and microbial test was attributed to the simplified model construction. The quality of processed oysters, including color, aroma and texture properties, was evaluated instrumentally by a digital image system, an electronic nose and universal testing machine. The performance of two electronic nose systems on their abilities to detect oyster aroma and classify the aroma data into distinct groups was evaluated using a trained sensory panel and microbial tests. Cyranose 320 system has demonstrated potential as a quality assessment tool due to its sound correlation with microbial quality data and sensory evaluation scores. According to the quality measurement results, high pressure processing conditions were recommended to be at 345 MPa for less than 3 min and 379 MPa for less than 1.5 min. Deterioration of the quality was distinct for oyster meats exposed to 60 °C or above by thermal processing. The critical thermal processing condition was identified to be 55 °C for 2 min. With careful control, microwave processing could be considered as a candidate for seafood processing to reduce potential bacterial hazard but still retain the quality of the product. / Ph. D.

No description available.

Live holding systems are used as temporary storage facilities for shellfish. The potential for mishandling of shellfish stored in these systems is high. The objective of the project was to examine the effects of storing oysters in a spray and an immersion systems on the survival of Escherichia coli and Vibrio vulnificus within the oysters. The effects of physiological stress imposed on oysters, as a result of interstate shipping, were examined by monitoring the level of E. coli in these oysters during storage in a spray tank. The survival rates of naturally-present E. coli and V. vulnificus in oysters were also observed. The research examined the distribution of artificially- and naturally-present V. vulnificus in oyster tissues during storage in an immersion system. There was no significant difference (p = 0.12) in the artificially-inoculated bacterial population of oysters after 120 hr of storage in a spray live holding tank. The level of E. coli in oysters which were subject to physiological stress did not change significantly (p = 0.30) after 96 hr in the spray tank. Naturally-present E. coli and V. vulnificus in oysters at harvest persisted during the 72 hr storage in the spray tank. V. vulnificus was loosely associated with mucus on the surfaces of the adductor and the mantle tissues in artificially-inoculated oysters. As a result, the bacterial level was reduced on these surfaces during the 72 hr of depuration. V. vulnificus on the gills and the digestive system of artificially-inoculated oysters may become entrapped in cilia and mucus. There was no significant reduction in the bacterial population on the gills (p = 0.11) and on the digestive system (p =0.21). There was no significant difference in the population of V. vulnificus in the adductor muscle (p = 0.37), the mantle (p = 0.16), the gills (p = 0.5), and the digestive system (p = 0.5) of summer oysters naturally-infected with the bacterium. It seems unlikely that depuration of V. vulnificus from oysters naturally harboring the bacterium may be effective. / Ph. D.

Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, <i>Vibrio fischeri</i>. In <i>V. fischeri</i>, the luminescence or <i>lux</i> genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the <i>lux</i> operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the <font face = "symbol">a</font>CTD of RNAP in LuxR-dependent transcriptional activation of the <i>lux</i> operon promoter has been investigated. The effect of seventy alanine substitution variants of the <font face = "symbol">a</font> subunit was determined <i>in vivo</i> by measuring the rate of transcription of the <i>lux</i> operon via luciferase assays in recombinant <i>Escherichia coli</i>. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by <i>in vitro</i> assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxR<font face = "symbol">D</font>N, was used for <i>in vitro</i> studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxR<font face = "symbol">D</font>N, and fourteen residues in the <font face = "symbol">a</font>CTD were identified as having negative effects on the rate of transcription from the <i>lux</i> operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxR<font face = "symbol">D</font>N-dependent activation <i>in vivo</i> and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type <font face = "symbol">a</font>CTD is capable of interacting with the <i>lux</i> DNA fragment tested, all five of the variant forms of the <font face = "symbol">a</font>CTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that <font face = "symbol">a</font>CTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the <i>lux</i> operon during quorum sensing. / Master of Science

Quorum sensing is utilized by many different proteobacteria, including the two studied for this dissertation work, Vibrio parahaemolyticus and Pantoea stewartii subsp. stewartii. V. parahaemolyticus causes acute gastroenteritis in people who eat contaminated raw or undercooked shellfish. It is found in warmer marine waters and in rare cases, causes systemic infections when bacteria enter the body through open wounds. P. stewartii, on the other hand, is a phytopathogen that causes Stewart's wilt in maize. It is found in soil or the mid-gut of the corn flea beetle, its insect vector. Both V. parahaemolyticus and P. stewartii utilize quorum sensing to control their pathogenicity. Quorum sensing enables coordinate gene expression across a bacterial population. The V. parahaemolyticus quorum-sensing system utilizes the master regulator OpaR, which is homologous to the V. harveyii LuxRVh and the P. stewartii system contains EsaR which is homologous to the V. fischeri LuxRVf regulator. While the two systems differ in the molecular details of their mechanistic control, they are both forms of cell density dependent regulation that are either directly or indirectly controlled by small signaling molecules. Three different signaling molecules are found in V. parahaemolyticus, and only one signal is used in P. stewartii. The focus of this dissertation has been on understanding the downstream targets of OpaR and EsaR in their respective quorum-sensing systems. Prior to this work, it was known that when OpaR is not present or is nonfunctional V. parahaemolyticus changes from an opaque to a translucent colony morphology phenotype and the cells also become swarm proficient and more pathogenic. The complete genome of the V. parahaemolyticus BB22OP strain was assembled and annotated (Chapter 2). RNA-Seq was then used to analyze the transcriptomes of OpaR-active and OpaR-deficient strains of V. parahaemolyticus and identify genes that were regulated via quorum sensing (Chapter 3). Similarly, P. stewartii was also analyzed using RNA-Seq to identify genes controlled by EsaR in the transcriptome that had not been detected through prior proteomic studies. The initial RNA-Seq work confirmed the control of some previously identified direct targets of EsaR and newly identified ten other genes also directly controlled by EsaR (Chapter 4). Two direct targets of EsaR, rcsA and lrhA, became the focus of additional studies to further define the hierarchy of gene control downstream of the quorum-sensing regulator EsaR. RcsA controls capsule production, while LrhA controls motility and adhesion in P. stewartii. The regulons of rcsA and lrhA were defined by RNA-Seq, which also revealed multi-level control of rcsA gene expression (Chapter 5). Tight coordinated and temporal control of virulence factors is important for successful disease progression by pathogens. This dissertation work aims to enable a better understanding of the quorum-sensing hierarchy of genetic control in V. parahaemolyticus and P. stewartii. / Ph. D.

Listeria monocytogenes, Vibrio parahaemolyticus and Morganella morganii are closely related to foodborne illnesses caused by the consumption of seafood and ready-to-eat (RTE) food. Traditional Chinese medicines (TCM) have been widely studied as complementary and alternative medicines, and many of them have been verified to have antimicrobial properties. The purpose of this research was to study antimicrobial effects of plant extracts as potential preservatives in seafood products and to identify the primary antimicrobial compounds in plant extracts. Four plants, Pomegranate peel (PP, Punica Granatum L.), Chinese gallnut (CG, Galla chinensis), forsythia fruit (FS, Forsythia suspensa) and Baikal skullcap root (BS, Scutellaria baicalensis) were ground and extracted with 70% methanol, respectively. The extracts were diluted at tested for antimicrobial activities on V. parahaemolyticus, L. monocytogenes and M. morganii both in agar diffusion assay using tryptic soy agar (TSA), and in microdilution assay using tryptic soy broth (TSB). Both CG and PP extracts, with concentrations no lower than 1 mg/ml, significantly inhibited both V. parahaemolyticus and L. monocytogenes (P<0.01) and reduced the bacterial population by up to 4 logs. No significant inhibition was observed with FS and BS extracts, except for BS at 5 mg/ml on V. parahaemolyticus. None of the extracts showed significant inhibition against M. morganii. The antibacterial activities of CG and PP 70% methanol extracts were tested in ground raw tuna and cooked tail-on shrimp. The extracts were mixed in tuna with final concentration at 1.7 mg/ml, and applied as soaking treatments (5 mg/ml) for shrimp. Both CG and PP extracts inhibited V. parahaemolyticus on both food matrices while only CG significantly inhibited L. monocytogenes. The 70% methanol crude extract of CG was analyzed by HPLC and LC-MS. Oligo-galloyl-O-glucose (nGG, n=1-10) are the major compounds in CG. The crude CG extract was fractionated using HPLC and the fractions were collected based on elution time and tested for their antimicrobial activities against V. parahaemolyticus and L. monocytogenes using agar diffusion methods. The fractions containing 3GG-8GG were the most active antimicrobials on both bacteria. / Ph. D.

Distributional ecology is a branch of ecology which aims to reconstruct and predict the geographic range of free-living and symbiotic organisms in terrestrial and aquatic ecosystems. More recently, distributional ecology has been used to map disease transmission risk. The implementation of distributional ecology for disease transmission has, however, been erroneous in many cases. The inaccurate representation of disease distribution is detrimental to effective control and prevention. Furthermore, ecological niche modeling experiments are generally developed and tested using data from terrestrial organisms, neglecting aquatic organisms in case studies. Both disease and aquatic systems are often data limited, and current modeling methods are often insufficient. There is, therefore, a need to develop data-driven models that perform accurately even when only limited amounts of data are available or when there is little to no knowledge of the species' natural history to be modeled. Here, I propose a data-driven ecological niche modeling method that requires presence-only data (i.e., absence, pseudoabsence, or background records are not needed for model calibration). My method is expected to reconstruct environmental conditions where data-limited aquatic organisms are more likely to be present, based on a density-based clustering algorithm as a proxy of the realized niche (i.e., abiotic, and biotic environmental conditions occupied by the organism). Supported by ecological theories and methods, my central hypothesis is that because density-based clustering machine-learning modeling prevents extrapolation and interpolation, it can robustly reconstruct the realized niche of a data-limited aquatic organism. First, I assembled a comprehensive dataset of abiotic (temperature) and biotic (phytoplankton) environmental conditions and presence reports using Vibrio cholerae, a well-understood aquatic bacterium species in coastal waters globally (Chapter 2). Second, using V. cholerae as a model system, I developed detailed parameterizations of density-based clustering models to determine the parameter values with the best capacities to reconstruct and predict the species' distribution in global seawaters (Chapter 3). Finally, I compared the performance of density-based clustering modeling against traditional, correlative machine-learning ecological niche modeling methods (Chapter 4). Density-based clustering models, when assessed based on model fit and prediction, had comparable performance to traditional 'data-hungry' machine-learning correlative methods used in modern applications of ecological niche modeling. Modeling the environmental and geographic ranges of V. cholerae, an aquatic organism of free-living and parasitic ecologies, is a novel approach itself in distributional ecology. Ecological niche modeling applications to pathogens, such as V. cholerae, provide an opportunity to further the knowledge of directly-transmitted emerging diseases for which only limited data are available. Density-based clustering ecological niche modeling is termed here as Marble, honoring a previous, experimental version of this analytical approach, and is expected to provide new opportunities to understand how an ecological niche modeling method influences estimates of the distribution of data-limited organisms of complex ecology. These are lessons applicable to novel, rare, and cryptic aquatic organisms, such as emerging diseases, endangered fishes, and elusive aquatic species. / Master of Science / Distributional ecology is a branch of ecology which aims to reconstruct and predict the geographic distribution of land and water organisms. In the case of diseases, a correct representation of their geographic distributions is key for successful management. Previous studies highlight the need to develop new models that perform accurately even when limited amounts of data are available and there is little to no knowledge of the organisms' ecology. This thesis proposes a data-driven method, originally termed Marble. Marble is expected to help reconstruct environmental conditions where data-limited aquatic organisms are more likely to be found. Supported by ecological theories and methods, my hypothesis is that because Marble prevents under- and over-fitting, this method will produce results which better fit the data. Using V. cholerae, an aquatic organism, as a model system, I compared the performance of Marble against other traditional modeling algorithms. I found that Marble, in terms of model fit, performed similarly to traditional methods used in distributional ecology. Modeling the ecology of V. cholerae is a new approach in and of itself in ecological modeling. Furthermore, modeling pathogens provides an opportunity to further the knowledge of directly transmitted diseases, and Marble is expected to provide opportunities to understand how algorithm selection can reconstruct (or not) the distribution of data-limited aquatic organisms of diverse ecologies.

The objective of this research was to determine if Vibrio populations, specifically V. vulnificus were affected in oysters by the processing methods employed in Virginia. This study was conducted between July and September in 1995 and during the month of August of 1996 when water temperature was expected to be high. Oysters were harvested from Virginia and the Gulf coast and shucked and blown by Virginia processors. They were tested for aerobic plate counts incubated at 35-37°C, salt content, pH, total Vibrios and V. vulnificus populations before and after processing. Oysters were stored in crushed ice and maintained an internal temperature of 1°C and tested at 5, 10, and 15 days after processing. Oysters were also stored at -9°C tested every one to two weeks. Procedures described in the Food and Drug Administration’s Bacteriological Analytical Manual for identification of V. vulnificus were followed. V. vulnificus populations were not significantly affected by blowing. V. vulnificus populations decreased in oysters stored at 1°C and -9°C. V. vulnificus levels decreased faster in blown oysters harvested from the Gulf coast. Vibrio populations were not significantly reduced by blowing in oysters that were 1°C. Oysters stored at -9°C showed decreased Vibrio populations. pH and APC showed an inverse relationship in oysters that were 1°C. In oysters stored at -9°C, pH and APC showed a positive correlation. Significance of these correlations varied. / Master of Science