Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27 degrees C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).     

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.     

Population structures of two genotypes of Vibrio vulnificus in oysters (Crassostrea virginica) and seawater

Vibrio vulnificus biotype 1 strains can be classified into two genotypes based on the PCR analysis of variations in the virulence-correlated gene (vcg). Genotype has been correlated with human infection for 90% of isolates from human cases having the vcgC sequence type and 87% of environmental strains having the vcgE variant. In this study we examined the dynamics of V. vulnificus populations and the distribution of the two genotypes recovered from oysters and surrounding estuarine wasters. Analysis of 880 isolates recovered from oysters showed a disparity in the ratio of the two genotypes, with those of the vcgE (E) genotype accounting for 84.4% of the population. In contrast, 292 isolates recovered from the waters surrounding the oyster sites revealed an almost equal distribution of the two genotypes. The levels of vcgC (C genotype) strains from both sources increased as a percentage of the population as water temperatures increased, while no culturable V. vulnificus cells were recovered from December through February. Our results suggest that there is a selective advantage for strains of the E genotype within oysters while survival of the C genotype strains may be favored by increased water column temperatures. These data suggest that the low incidence of infections may be due to the comparatively rare consumption of an oyster that contains a greater number of V. vulnificus vcgC genotype strains than of vcgE genotype strains. Levels of the two genotypes as well as seasonal dynamics within both oyster tissue and the surrounding waters may aid in identifying risk factors associated with human infection.     

Densities of Vibrio vulnificus in the intestines of fish from the U.S. Gulf Coast.

Densities of Vibrio vulnificus in the intestinal contents of various finfish, oysters, and crabs and in sediment and waters of the U.S. Gulf Coast were determined by the most probable number procedure. Species were identified by enzyme immunoassay. During the winter, densities of V. vulnificus were low, and the organism was isolated more frequently from sheepshead fish than from sediment and seawater. From April to October, V. vulnificus densities were considerably higher (2 to 5 logs) in estuarine fish than in surrounding water, sediment, or nearby oysters and crustacea. Highest densities were found in the intestinal contents of certain bottom-feeding fish (10(8)/100 g), particularly those that consume mollusks and crustaceans. Densities of V. vulnificus in fish that feed primarily on plankton and other finfish were similar to those in oysters, sediment, and crabs (10(5)/100 g). V. vulnificus was found infrequently in offshore fish. The presence of high densities of V. vulnificus in the intestines of common estuarine fish may have both ecological (growth and transport) and public health (food and wound infections) implications.     

Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats.

Phages lytic to Vibrio vulnificus were found in estuarine waters, sediments, plankton, crustacea, molluscan shellfish, and the intestines of finfish of the U.S. Gulf Coast, but no apparent relationship between densities of V. vulnificus and its phages was observed. Phage diversity and abundance in molluscan shellfish were much greater than in other habitats. V. vulnificus phages isolated from oysters did not lyse other mesophilic bacteria also isolated from oysters. Both V. vulnificus and its phages were found in a variety of oyster tissues and fluids with lowest densities in the hemolymph and mantle fluid. These findings suggest a close ecological relationship between V. vulnificus phages and molluscan shellfish.     

Temporal and Spatial Variability in the Distribution of Vibrio vulnificus in the Chesapeake Bay: A Hindcast Study

Vibrio vulnificus, an estuarine bacterium, is the causative agent of seafood-related gastroenteritis, primary septicemia, and wound infections worldwide. It occurs as part of the normal microflora of coastal marine environments and can be isolated from water, sediment, and oysters. Hindcast prediction was undertaken to determine spatial and temporal variability in the likelihood of occurrence of V. vulnificus in surface waters of the Chesapeake Bay. Hindcast predictions were achieved by forcing a multivariate habitat suitability model with simulated sea surface temperature and salinity in the Bay for the period between 1991 and 2005 and the potential hotspots of occurrence of V. vulnificus in the Chesapeake Bay were identified. The likelihood of occurrence of V. vulnificus during high and low rainfall years was analyzed. From results of the study, it is concluded that hindcast prediction yields an improved understanding of environmental conditions associated with occurrence of V. vulnificus in the Chesapeake Bay.     

The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina Estuaries

While numerous studies have characterized the distribution and/or ecology of various pathogenic Vibrio spp., here we have simultaneously examined several estuarine sites for Vibrio vulnificus, V. cholerae, and V. parahaemolyticus. For a one year period, waters and sediment were monitored for the presence of these three pathogens at six different sites on the east coast of North Carolina in the United States. All three pathogens, identified using colony hybridization and PCR methods, occurred in these estuarine environments, although V. cholerae occurred only infrequently and at very low levels. Seventeen chemical, physical, and biological parameters were investigated, including salinity, water temperature, turbidity, dissolved oxygen, levels of various inorganic nutrients and dissolved organic carbon, as well as total vibrios, total coliforms, and E. coli. We found each of the Vibrio spp. in water and sediment to correlate to several of these environmental measurements, with water temperature and total Vibrio levels correlating highly (P<0.0001) with occurrence of the three pathogens. Thus, these two parameters may represent simple assays for characterizing the potential public health hazard of estuarine waters.     

Intraspecific diversity of Vibrio vulnificus in Galveston Bay water and oysters as determined by randomly amplified polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

The reductase component of the chromosomally encoded benzoate dioxygenase from Pseudomonas putida C-1 is immunologically homologous with a product of the plasmid encoded xyl D gene (toluate dioxygenase) from Pseudomonas putida mt-2

Vibrio vulnificus, an autochthonous inhabitant of the estuarine environment, was detected in water and oysters from the Great Bay Estuary System of New Hampshire and Maine. Previously, it had not been detected north of Boston Harbor on the east coast of the United States. V. vulnificus was detected in water and shellfish samples at five out of ten sites, and only in areas that were not open to recreational shellfishing. Although samples were collected from May into December, V. vulnificus was only detected in shellfish in July and August. Water sampling began in August, and V. vulnificus persisted at one site into October.     

Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus

The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene (toxR) of Vibrio vulnificus. The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus. We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.     

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 (10(2) to 10(8) CFU ml(-1)), with a lower limit of 72 fg of genomic DNA micro l 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 (r(2) = 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.     

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.     

Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review

The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat to humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification

Vibrio vulnificus is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmental V. vulnificus strains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study, vcgC was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulent V. vulnificus strains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulent V. vulnificus strain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 10(3) CFU/g of V. vulnificus ATCC 33815, while showing negative results for a nonvirulent V. vulnificus strain (515-4c2) spiked at 10(7) CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulent V. vulnificus strain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulent V. vulnificus strain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulent V. vulnificus in raw oysters with high speed, specificity, and sensitivity, which may facilitate better control of V. vulnificus risks associated with raw oyster consumption.     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

Lethal cold stress of Vibrio vulnificus in oysters.

Studies were conducted on the survival of Vibrio vulnificus, an estuarine human pathogen, in oyster homogenates held at 4 degrees C. Results indicated a rapid and dramatic decrease in viability not attributable to either cold shock or the oyster homogenate alone but to a combination of the two. Such a decline was not observed with Vibrio parahaemolyticus. Chilled V. vulnificus cells were unable to repair themselves in brain heart infusion broth at 37 degrees C. V. vulnificus cells incubated on whole raw oysters at 0.5 degrees C also exhibited a decline in viability, but of a lesser degree. The effects of various plating media were also investigated. The data reported here suggest that oysters kept on ice are not likely to be a major factor in the epidemiology of V. vulnificus infection. It is further suggested that the standard method of homogenizing oysters for examining bacteriological quality should not be followed because toxic compounds are released from the oysters during this process.     

Occurrence of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.     

Viability of Vibrio vulnificus in Association with Hemocytes of the American Oyster (Crassostrea virginica)

Certain indigenous estuarine bacteria, such as Vibrio vulnificus, may cause opportunistic human infections after consumption of raw oysters or exposure of tissues to seawater. V. vulnificus is known to be closely associated with oyster (Crassostrea virginica) tissues and is not removed by controlled purification methods, such as UV light-assisted depuration. In fact, when live shellfish are subjected to controlled purification, the number of V. vulnificus cells can markedly increase. A review of previous studies showed that few workers have examined mechanisms in oysters which may influence the persistence of V. vulnificus in shellfish, such as the fate of V. vulnificus following phagocytosis by molluscan hemocytes. The objectives of this study were to define the intracellular viability and extracellular viability of V. vulnificus during the phagocytic process and to study the release of specific lysosomal enzymes. The viability of a virulent estuarine V. vulnificus isolate with opaque morphology was compared with the viability of a translucent, nonvirulent form, the viability of Vibrio cholerae, and the viability of Escherichia coli in phagocytosis experiments. Our results showed that the levels of phagocytosis and bactericidal degradation of the opaque V. vulnificus isolate were less than the levels of phagocytosis and bactericial degradation of the translucent morphotype. These findings indicate that encapsulation may contribute to resistance to ingestion and degradation by hemocytes. The rates of intracellular death of V. cholerae and E. coli exceeded the rate of intracellular death of the opaque V. vulnificus isolate, even though the ingestion or uptake rates did not differ significantly. The levels of lysozyme activity and acid phosphatase activity were not significantly different in hemocyte monolayers inoculated with V. vulnificus.