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.     

The Interactions of Vibrio vulnificus and the Oyster Crassostrea virginica

The human bacterial pathogen, Vibrio vulnificus, is found in brackish waters and is concentrated by filter-feeding molluscan shellfish, especially oysters, which inhabit those waters. Ingestion of raw or undercooked oysters containing virulent strains of V. vulnificus can result in rapid septicemia and death in 50 % of victims. This review summarizes the current knowledge of the environmental interactions between these two organisms, including the effects of salinity and temperature on colonization, uptake, and depuration rates of various phenotypes and genotypes of the bacterium, and host–microbe immunological interactions.     

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.     

Copper exposure affects hemocyte apoptosis and Perkinsus marinus infection in eastern oysters Crassostrea virginica (Gmelin)

Dermo disease in the eastern oyster (Crassostrea virginica) is caused by an intracellular protistan parasite Perkinsus marinus. The progression and outcome of this disease is determined by a complex interplay between the host's immunity and parasite's escape mechanisms, both of which can be influenced by environmental pollutants including heavy metals such as copper (Cu). The goal of the present study was to determine the effects of Cu on the levels of apoptosis (which can serve as an important host defense mechanism) in oyster immune cells (hemocytes) in?vitro and in?vivo as well as on the establishment of P.?marinus infections in?vivo. Surprisingly, Cu exerted opposing effects on apoptosis levels of hemocytes in?vitro and in?vivo, stimulating apoptosis in isolated hemocytes but suppressing it during Cu exposure of whole oysters. The mechanisms of this effect are presently unknown and may be related to the different bioavailability of the metal in?vitro and in?vivo. As expected, Cu accumulated in oyster soft tissues during in?vitro exposure. Unexpectedly, this metal also strongly accumulated in hemolymph plasma which is classically considered isoionic with the surrounding seawater, likely reflecting the presence of soluble Cu-binding proteins in oyster plasma. Cu reduced growth of P.?marinus in?vitro and greatly reduced infection levels of hemocytes in?vivo, presumably by direct toxic effects on the parasite. As a possible parasitic counterbalance, Cu accumulation in the hemocytes was reduced by P.?marinus infection, although this reduction was not sufficient to prevent the parasiticidal effects of the heavy metal in?vivo. This effect of Cu may be useful as a potential therapeutic against Dermo disease in aquaculture conditions. Overall, this study provides important new insights into the potential role of environmental metals in host-parasite relationships and disease dynamics in C.?virginica.     

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.     

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.     

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica)

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Bacteriophage Clearance in the Oyster (Crassostrea virginica)

Since relatively few quantitative immunological studies had been conducted with invertebrates, this study was designed to learn more about the immune potential of the oyster, a molluscan representative. Experiments measured the primary and secondary clearance rates of T2 coliphage in the oyster. The fate of phage injected intracardially or intramuscularly was traced by measuring serum and tissue fluid plaque-forming unit levels at various times postinjection. Phage-neutralizing activity was also measured. Although circulating T2-neutralizing antibody was not induced in the oyster, secondary injections of T2 were cleared more rapidly than primary injections. The difference in clearance rates between primary and secondary groups was not as pronounced in the oyster as in the lemon shark, an animal capable of producing high levels of serum antibody to T2.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica)

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Characterization of a Marine Bacterium Associated with Crassostrea virginica (the Eastern Oyster)

A gram-negative bacterium found to be closely associated with oysters has been isolated and characterized. The organism, designated LST, has a generation time of 106 min in Marine broth under optimal growth conditions at 25°C. During the decline phase of growth, it exhibits a morphological transition from a motile rod (ca. 1 μm in length) to an elongated, 3- to 40-μm, nonmotile, tightly coiled helix. LST synthesizes and releases a pigment in the stationary and decline phases of growth. Identified as melanin on the basis of chemical properties and UV absorbance maxima, the pigment comprises polymers of heterogeneous molecular weights, ranging from 12,000 to 120,000. The guanosine-plus-cytosine content of the LST DNA is 46%, and results of phenetic analysis and DNA-DNA hybridization indicate that this bacterium represents a new species. LST adheres to a variety of surfaces, including glass, plastics, and oyster shell, and has been shown to promote the settlement of oyster larvae.     

Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica).

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.     

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.     

Uptake and clearance of Vibrio vulnificus from Gulf coast oysters (Crassostrea virginica).

Oysters collected in late winter, when they were free of Vibrio vulnificus, were exposed in the organism in the laboratory. The oysters effectively concentrated the bacteria from seawater, but when the inoculum was removed, the bacteria were rapidly cleared from the oyster tissues. These results suggest that V. vulnificus may be found in oysters as a result of filtration of the bacteria from seawater rather than active multiplication of the bacteria in the oysters.     

Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica).

Oysters naturally contaminated with 10(3) to 10(4) most probable numbers (MPN) of Vibrio vulnificus per g were relayed to offshore waters (salinity, 30 to 34 ppt), where they were suspended in racks at a depth of 7.6 m. V. vulnificus counts in oysters were reduced to < 10 MPN/g within 7 to 17 days in five of the six studies. At the end of the studies (17 to 49 days), V. vulnificus levels were reduced further and ranged from a mean of 0.23 to 2.6 MPN/g. Oyster mortalities during relaying were < 6%. The reduction of V. vulnificus in relayed oysters is associated with exposure to high-salinity environments essentially devoid of V. vulnificus. Offshore suspension relaying may be a method that industry can employ to reduce V. vulnificus levels in raw Gulf Coast oysters.