Effects of Physicochemical Factors and Bacterial Colony Morphotype on Association of Vibrio vulnificus with Hemocytes of Crassostrea virginica

Vibrio vulnificus is a naturally occurring marine bacterium that causes invasive disease of immunocompromised humans following the consumption of raw oysters. It is a component of the natural microbiota of Gulf Coast estuaries and has been found to inhabit tissues of oysters, Crassostrea virginica (Gmelin 1791). The interaction of V. vulnificus with oyster host defenses has not been reported in detail. We examined the interaction of V. vulnificus with phagocytic oyster hemocytes as a function of time, temperature, bacterial concentration, pretreatment with hemolymph, and V. vulnificus translucent and opaque colony morphotypes. Within these experimental parameters, the results showed that the association of V. vulnificus with hemocytes increased with time, temperature, and initial V. vulnificus/hemocyte ratio. Pretreatment of V. vulnificus with serum or an increased serum concentration did not enhance V. vulnificus-hemocyte associations, a result suggesting the absence of opsonic activity. More than 50% of hemocytes bound the translucent, avirulent morphotype, whereas 10 to 20% were associated with the opaque, virulent form, a result indicating that the degree of encapsulation was related to resistance to phagocytosis, as previously described for mammalian phagocytes. Understanding these cellular interactions may, in part, explain the persistence of V. vulnificus in oyster tissues and the ecology of V. vulnificus in estuarine environments.     

Factors Influencing In Vitro Killing of Bacteria by Hemocytes of the Eastern Oyster (Crassostrea virginica)

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster’s cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp,Fenneropenaeus chinensis

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.     

Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10² V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.     

Supplementation of Perkinsus marinus Cultures with Host Plasma or Tissue Homogenate Enhances Their Infectivity

The protozoan oyster parasite Perkinsus marinus can be cultured in vitro in a variety of media; however, this has been associated with a rapid attenuation of infectivity. Supplementation of defined media with products of P. marinus-susceptible (Crassostrea virginica) and -tolerant (Crassostrea gigas, Crassostrea ariakensis) oysters alters proliferation and protease expression profiles and induces differentiation into morphological forms typically seen in vivo. It was not known if attenuation could be reversed by host extract supplementation. To investigate correlations among these changes as well as their association with infectivity, the effects of medium supplementation with tissue homogenates from both susceptible and tolerant oyster species were examined. The supplements markedly altered both cell size and proliferation, regardless of species; however, upregulation of low-molecular-weight protease expression was most prominent with susceptible oysters extracts. Increased infectivity occurred with the use of oyster product-supplemented media, but it was not consistently associated with changes in cell size, cell morphology, or protease secretion and was not related to the susceptibility of the oyster species used as the supplement source.     

Sediment and Vegetation as Reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.     

Real-Time PCR Analysis of Vibrio vulnificus from Oysters

Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (10² to 10⁸ CFU ml⁻¹), with a lower limit of 72 fg of genomic DNA μl of PCR mixture⁻¹ or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r² = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.     

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C_T) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10³ V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 10³ CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

Hemocytes and Plasma of the Eastern Oyster (Crassostrea virginica) Display a Diverse Repertoire of Sulfated and Blood Group A-modified N-Glycans

The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1.     

Regulation systems of protease and hemolysin production in Vibrio vulnificus

Vibrio vulnificus, a gram‐negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema‐forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone‐like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.     

Influence of Water Temperature and Salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast Oysters (Crassostrea virginica)

This study investigated the temperature and salinity parameters associated with waters and oysters linked to food-borne Vibrio vulnificus infections. V. vulnificus was enumerated in oysters collected at three northern Gulf Coast sites and two Atlantic Coast sites from July 1994 through September 1995. Two of these sites, Black Bay, La., and Apalachicola Bay, Fla., are the source of the majority of the oysters implicated in V. vulnificus cases. Oysters in all Gulf Coast sites exhibited a similar seasonal distribution of V. vulnificus: a consistently large number (median concentration, 2,300 organisms [most probable number] per g of oyster meat) from May through October followed by a gradual reduction during November and December to ≤10 per g, where it remained from January through mid-March, and a sharp increase in late March and April to summer levels. V. vulnificus was undetectable (<3 per g) in oysters from the North and South Carolina sites for most of the year. An exception occurred when a late-summer flood caused a drop in salinity in the North Carolina estuary, apparently causing V. vulnificus numbers to increase briefly to Gulf Coast levels. At Gulf Coast sites, V. vulnificus numbers increased with water temperatures up to 26°C and were constant at higher temperatures. High V. vulnificus levels (>10³ per g) were typically found in oysters from intermediate salinities (5 to 25 ppt). Smaller V. vulnificus numbers (<10² per g) were found at salinities above 28 ppt, typical of Atlantic Coast sites. On 11 occasions oysters were sampled at times and locations near the source of oysters implicated in 13 V. vulnificus cases; the V. vulnificus levels and environmental parameters associated with these samples were consistent with those of other study samples collected from the Gulf Coast from April through November. These findings suggest that the hazard of V. vulnificus infection is not limited to brief periods of unusual abundance of V. vulnificus in Gulf Coast oysters or to environmental conditions that are unusual to Gulf Coast estuaries.     

Application of Chitosan Microparticles for Reduction of Vibrio Species in Seawater and Live Oysters (Crassostrea virginica)

Human Vibrio infections associated with consumption of raw shellfish greatly impact the seafood industry. Vibrio cholerae-related disease is occasionally attributed to seafood, but V. vulnificus and V. parahaemolyticus are the primary targets of postharvest processing (PHP) efforts in the United States, as they pose the greatest threat to the industry. Most successful PHP treatments for Vibrio reduction also kill the molluscs and are not suitable for the lucrative half-shell market, while nonlethal practices are generally less effective. Therefore, novel intervention strategies for Vibrio reduction are needed for live oyster products. Chitosan is a bioactive derivative of chitin that is generally recognized as safe as a food additive by the FDA, and chitosan microparticles (CMs) were investigated in the present study as a potential PHP treatment for live oyster applications. Treatment of broth cultures with 0.5% (wt/vol) CMs resulted in growth cessation of V. cholerae, V. vulnificus, and V. parahaemolyticus, reducing culturable levels to nondetectable amounts after 3 h in three independent experiments. Furthermore, a similar treatment in artificial seawater at 4, 25, and 37°C reduced V. vulnificus levels by ca. 7 log CFU/ml after 24 h of exposure, but 48 h of exposure and elevated temperature were required to achieve similar results for V. parahaemolyticus and V. cholerae. Live oysters that either were artificially inoculated or contained natural populations of V. vulnificus and V. parahaemolyticus showed significant and consistent reductions following CM treatment (5%) compared to the amounts in the untreated controls. Thus, the results strongly support the promising potential for the application of CMs as a PHP treatment to reduce Vibrio spp. in intact live oysters.     

Refined Medium for Direct Isolation of Vibrio vulnificus from Oyster Tissue and Seawater

We have developed a new medium for the direct isolation of Vibrio vulnificus from water and oyster samples. The medium was shown in laboratory and field studies to be highly selective without providing preferential isolation of either V. vulnificus genotype.     

Abundance of Vibrio cholerae,V. vulnificus,and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh⁺ and/or trh⁺) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.     

Chemotactic attraction between hemocytes of the oyster,Crassostrea virginica,and bacteria

Experiments were conducted to ascertain whether there is chemotactic attraction by Bacillus megaterium and Micrococcus varians, both Gram-positive species, and Escherichia coli and Vibrio parahaemolyticus, both Gram-negative species, for hemocytes of the American oyster, Crassostrea virginica. It was ascertained quantitatively that oyster hemocytes are attracted to live E. coli, B. megaterium, and M. varians but not to heat-killed bacteria. Furthermore, oyster cells are not attracted to either live or heat-killed V. parahaemolyticus. It is concluded that the chemoattractant is some molecule emitted by living vegetative cells of certain Gram-positive as well as Gramnegative bacteria.     

Validation of a Green Fluorescent Protein-Labeled Strain of Vibrio vulnificus for Use in the Evaluation of Postharvest Strategies for Handling of Raw Oysters

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45°C), freeze-thaw tolerance (−20^o and −80°C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5°C), cold adaptation (15°C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log₁₀ after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10² CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10³ and >10⁴ CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.     

Development of a Matrix Tool for the Prediction of Vibrio Species in Oysters Harvested from North Carolina

The United States has federal regulations in place to reduce the risk of seafood-related infection caused by the estuarine bacteria Vibrio vulnificus and Vibrio parahaemolyticus. However, data to support the development of regulations have been generated in a very few specific regions of the nation. More regionally specific data are needed to further understand the dynamics of human infection relating to shellfish-harvesting conditions in other areas. In this study, oysters and water were collected from four oyster harvest sites in North Carolina over an 11-month period. Samples were analyzed for the abundances of total Vibrio spp., V. vulnificus, and V. parahaemolyticus; environmental parameters, including salinity, water temperature, wind velocity, and precipitation, were also measured simultaneously. By utilizing these data, preliminary predictive management tools for estimating the abundance of V. vulnificus bacteria in shellfish were developed. This work highlights the need for further research to elucidate the full suite of factors that drive V. parahaemolyticus abundance.