Oysters are filter-feeding bivalves, which filter water for nutrients and often accumulate contaminants and human pathogens such as Vibrio parahaemolyticus and Vibrio vulnificus naturally occurring in the marine environment. These naturally occurring pathogens have been frequently isolated from raw shellfish, particularly oyster, in the United States and are recognized as the leading causes of human gastroenteritis associated with seafood consumption. Human illness caused by consumption of raw oyster contaminated with V. parahaemolyticus and Vibrio vulnificus typically results in reduced sales of oysters and a consequent significant financial burden for the producers. The United States produces more than 27 million pounds of oysters each year with a large portion of them being produced from the coastal water of the Gulf of Mexico. It is estimated that 20 million Americans eat raw shellfish and consumption of raw oyster is responsible for about 95% of all deaths associated with seafood consumption in the U.S., making raw oysters one of the most hazardous seafoods. Several post-harvest processes, including low temperature pasteurization, freezing, high pressure processing and irradiation, have been reported capable of reducing Vibrio contamination in raw oysters. However, most of them require either a significant amount of initial investment or operation costs, and oysters are often killed during processing. Cost-effective post-harvest processing for reducing V. parahaemolyticus in raw oysters without significant adverse effects on the oysters remains to be developed. This study was conducted to determine impacts of low-temperature (15, 10 and 5°C) depuration and frozen storage on reducing V. parahaemolyticus and V. vulnificus in raw oysters. Depuration of the Gulf oyster (Crassostrea virginica) with electrolyzed oxidizing (EO) water (chlorine, 30 ppm; pH 2.82; oxidation-reduction potential, 1,131mV) containing 3% NaCl was found ineffective on reducing both V. parahaemolyticus and V. vulnificus in the oysters. Reductions of V. parahaemolyticus and V. vulnificus in oyster after 48 h of EO water depuration at 22°C were limited to 0.7 and 1.4 log MPN/g, respectively. Depuration with EO water at lower temperatures did not enhance reductions of Vibrio in the oysters. Greater reductions of V. parahaemolyticus (1.2 log MPN/g) and V. vulnificus (2.0 log MPN/g) were observed when the oysters were depurated with artificial seawater (ASW) at room temperature (22°C) for 48 h. Decreasing temperature of ASW to 15°C for depuration significantly increased the reductions of V. parahaemolyticus and V. vulnificus to 2.1 and 2.9 log MPN/g, respectively, after 48 h of process. However, depuration of oyster in ASW at 10 and 5°C were found less effective than at 15°C in reducing Vibrio in the Gulf oysters. An extended depuration with ASW at 15°C for 96 h was capable of achieving 2.6 and 3.3 log MPN/g of reductions of V. parahaemolyticus and V. vulnificus, respectively, in the Gulf oysters. Study of effects of frozen storage at -10, -23 and -30°C on reducing V. parahaemolyticus in raw half-shell Pacific oyster (Crassostrea gigas) found that the population of the bacterium decreased faster in oysters stored at -10 than at -23 or -30°C. Holding half-shell Pacific oyster at -10°C for three months or at -23°C for four months was capable of achieving a greater than 3-log (MPN/g) reduction of V. parahaemolyticus in the Pacific oyster. / Graduation date: 2008

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.

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.

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

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Gulf oysters are harvested and consumed year-round, with more than 90% consumed in a raw, unprocessed state. A chief concern of policymakers in recent years is the incidence of Vibrio vulnificus infection following raw seafood consumption. V.vulnificus refers to a halophilic bacterium naturally occurring in brackish coastal waters, which concentrates in filter-feeding oysters. Proposed FDA legislation requiring processing of all raw Gulf oysters sold during warmer summer months threatens the Gulf oyster industry, as little to no research regarding demand for post-harvest processing (PHP) has preceded the potential mandate. This research endeavors to examine the relationship between oyster consumers' fears of V.vulnificus infection and their willingness-to-pay (WTP) for processing of an oyster meal. The psychological model of Protection Motivation Theory (PMT) is employed alongside the economic framework of contingent valuation (CV) to result in an analysis of oyster processing demand with respect to threats and efficacy. A survey administered to 2,172 oyster consumers in six oyster producing states elicits projected consumption and PMT data. Principal Component Analysis is used to reduce the number of PMT variables to a smaller size, resulting in five individual principal components representing the PMT elements of source information, threat appraisal, coping appraisal, maladaptive coping, and protection motivation. Using survey data, the marginal willingness-to-pay (MWTP) for PHP per oyster meal is also calculated, and the five created PMT variables are regressed on this calculation using four separate OLS models. Results indicate significant correlation for four of the five created PMT variables. In addition, a mean MWTP for PHP of $0.31 per oyster meal is determined, contributing to the demand analysis for processing of Gulf oysters. The findings suggest a strong relationship between the fear elements and the demand for processing, and support arguments in favor of further research on specific PHP treatments and the necessity for a valid PMT survey instrument.

[ES] La tesis doctoral "Nanostructured supports for the detection of pathogens and biomolecules of interest based on molecular gates with oligonucleotides" se centra en el diseño de nanomateriales híbridos orgánico-inorgánicos como biosensores innovadores para la detección rápida y específica de patógenos como el virus del papiloma humano (VPH) y la bacteria Vibrio vulnificus, así como biomoléculas relevantes como miR-4732-3p, relacionado con la cardiotoxicidad en pacientes con cáncer. Los sensores desarrollados, basados en puertas moleculares en alúmina mesoporosa y validados con muestras clínicas, destacan por su alta sensibilidad, especificidad, rapidez y facilidad de uso, lo que los hace prometedores para aplicaciones en diagnóstico y monitoreo ambiental. / [CA] The doctoral thesis "Nanostructured supports for the detection of pathogens and biomolecules of interest based on molecular gates with oligonucleotides" focuses on the design of organic-hybrid nanomaterialsInorganic as innovative biosensors for rapid and specific detection of pathogens such as human papillomavirus (HPV) and the bacterium Vibrio vulnificus, as well as relevant biomolecules such as miR-4732-3p, related to cardiotoxicity in cancer patients. The developed sensors, based on mesoporous alumina molecular gates and validated with clinical samples, stand out for their high sensitivity, specificity, speed and ease of use, which makes them promising for applications in diagnosis and environmental monitoring. / [EN] La tesi doctoral "Nanostructured supports for the detection of pathogens and biomolecules of interest based on molecular gates with oligonucleotides" se centra en el disseny de nanomaterials híbrids orgànic-inorgànics com biosensors innovadors per a la detecció ràpida i específica de patògens com el virus del papil·loma humà (VPH) i el bacteri Vibrio vulnificus, així com biomolècules rellevants com miR-4732-3p, relacionat amb la cardiotoxicitat en pacients amb càncer. Els sensors desenrotllats, basats en portes moleculars en alúmina mesoporosa i validats amb mostres clíniques, destaquen per la seua alta sensibilitat, especificitat, rapidesa i facilitat d'ús, la qual cosa els fa prometedors per a aplicacions en diagnòstic i monitoratge ambiental. / This research was supported by project PID2021-126304OB-C41 funded by MCIN/AEI and by European Regional Development Fund - A way of doing Europe. This work has received funding from the European Union’s Horizon 2020 Development of an Innovative Fluorogenic Biosensor for Direct Detection of Vibrio vulnificus, a Climate Change Biomarker research and innovation programme under grant agreement No 899708. This research was also supported by CIBER -Consorcio Centro de Investigación Biomédica en Red (CB06/01/2012), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. This study forms part of the Advanced Materials programme (MFA/2022/049) and was supported by MCIN with funding from European Union NextGeneration EU (PRTR-C17.I1) and by Generalitat Valenciana. The study was also supported by the grants PID2020-120619RB-I00 funded by MCIN/AEI and CIAICO/2021/293 funded by “Conselleria de Educación, Universidades y Empleo” (Generalitat Valenciana, Spain). / Aranda Sobrino, MDLN. (2024). Nanostructured Supports for Detection of Pathogens and Biomolecules of Interest Based on Molecular Gates with Oligonucleotides [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/211237