A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD.     

Epizootiology and pathology of juvenile oyster disease in the Eastern oyster, Crassostrea virginica.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60-90% in oysters <25 mm. Mantle lesions were highly correlated with subsequent conchiolin-deposit prevalence and with total mortality. Larger juveniles (25-40 mm) were affected by the disease and produced conchiolin deposits, but mortalities did not exceed 30%. Mortalities were consistently related to size, but not necessarily to age or length of "exposure" in the field. There was no indication that JOD was linked to a particular broodstock or hatchery. Wild spat deployed at experimental sites showed JOD symptoms before the hatchery-produced groups did and cohorts maintained inside a hatchery experienced essentially no JOD. Histological examination of cohorts experiencing high mortalities failed to reveal an obvious etiological agent, but showed a disease pattern similar to that described for other bivalve diseases with a bacterial etiology. Similarities and differences between this and other studies of JOD suggest that one or more bacterial species is responsible for JOD, but that a trigger, probably temperature, is also involved and may vary from site to site.     

Localization of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected eastern oysters (Crassostrea virginica)

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters (Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.     

Esterification of vertebrate-type steroids in the Eastern oyster (Crassostrea virginica)

Characteristics of acyl-coenzyme A (acyl-CoA):steroid acyltransferase from the digestive gland of the oyster Crassostrea virginica were determined by using estradiol (E2) and dehydroepiandrosterone (DHEA) as substrates. The apparent Km and Vmax values for esterification of E2 with the six fatty acid acyl-CoAs tested (C20:4, C18:2, C18:1, C16:1, C18:0, and C16:0) were in the range of 9-17 microM E2 and 35-74 pmol/min/mg protein, respectively. Kinetic parameters for esterification of DHEA (Km: 45-120 microM; Vmax: 30-182 pmol/min/mg protein) showed a lower affinity of the enzyme for this steroid. Formation of endogenous fatty acid esters of steroids by microsomes of digestive gland and gonads incubated in the presence of ATP and CoA was assessed, and at least seven E2 fatty acid esters and five DHEA fatty acid esters were observed. Some peaks eluted at the same retention times as palmitoleoyl-, linoleoyl-, oleoyl/palmitoyl-, and stearoyl-E2; and palmitoleoyl-, oleoyl/palmitoyl-, and stearoyl-DHEA. The same endogenous esters, although in different proportions, were produced by gonadal microsomes. The kinetic parameters for both E2 (Km: 10 microM; Vmax: 38 pmol/min/mg protein) and DHEA (Km: 61 microM; Vmax: 60 pmol/min/mg protein) were similar to those obtained in the digestive gland. Kinetic parameters obtained are similar to those observed in mammals; thus, fatty acid esterification of sex steroids appears to be a well-conserved conjugation pathway during evolution.     

Chronic infections of Haplosporidium nelsoni (MSX) in the oyster Crassostrea virginica

Oysters inhabiting areas enzootic for the parasite Haplosporidium nelsoni (MSX) are exposed to infective particles each summer, and it is often difficult to distinguish newly acquired lesions from older infections. To study long-term parasitism without the complication of new infections, MSX-infected oysters were moved from Delaware Bay to a disease-free area on the New Jersey coast. Because infections seen after the transfer were acquired in Delaware Bay during a known infective period, it was possible to determine how long oysters remained infected and how they were affected by chronic parasitism. Chronic infections displayed the same seasonal cycle (high levels in winter and late spring, low levels in summer and early spring) that occurs in enzootic areas with annual reinfection. Within individual oysters, chronic MSX became localized, relapsed into general infections, and then became localized again in a sequence that was probably controlled by temperature. Some experimental oysters survived with MSX for at least 4 years. Hemocytosis persisted in these oysters, and their poor condition suggested that chronically infected individuals would have lowered resistance to additional stress.     

Use of Antibacterial Agents To Elucidate the Etiology of Juvenile Oyster Disease (JOD) in Crassostrea virginica and Numerical Dominance of an α-Proteobacterium in JOD-Affected Animals

Since 1988, juvenile oyster disease (JOD) has resulted in high seasonal losses of cultured Eastern oysters (Crassostrea virginica) in the Northeast. Although the cause of JOD remains unknown, most evidence is consistent with either a bacterial or a protistan etiology. For the purpose of discerning between these hypotheses, the antibacterial antibiotics norfloxacin and sulfadimethoxine-ormetoprim (Romet-B) were tested for the ability to delay the onset of JOD mortality and/or reduce the JOD mortality of cultured juvenile C. virginica. Hatchery-produced C. virginica seed were exposed in triplicate groups of 3,000 animals each to either norfloxacin, sulfadimethoxine-ormetoprim, or filter-sterilized seawater (FSSW) and deployed in floating trays on the Damariscotta River of Maine on 17 July 1997. Each week thereafter, a subset of animals from each group was reexposed to the assigned treatment. Repeated immersion in either a sulfadimethoxine-ormetoprim or a norfloxacin solution resulted in a delay in the onset of JOD mortality in treated animals and reduced weekly mortality rates. Weekly treatments with either norfloxacin or sulfadimethoxine-ormetoprim also resulted in a statistically significant reduction in cumulative mortality (55 and 67% respectively) compared to animals treated weekly with FSSW (81%) or those that had received only a single treatment with either norfloxacin, sulfadimethoxine-ormetoprim, or FSSW (77, 84, and 82%, respectively). Bacteriological analyses revealed a numerically dominant bacterium in those animals with obvious signs of JOD. Sequence analysis of the 16S rRNA gene from these bacteria indicates that they are a previously undescribed species of marine α-proteobacteria.     

A lectin on the hemocyte membrane of the oyster (Crassostrea virginica)

Using antisera produced against a serum lectin we have shown by employing immunocytofluorescence that hemocytes from the oyster, Crassostrea virginica, possess a lectin which is situated on the external surface of the cell membrane. The antisera block the binding of hemocyte microsomes to protease-treated vertebrate erythrocytes, thus confirming that the hemocyte membrane lectin is serologically related to the serum lectin. The major serum lectin has an apparent mass of 34,000. Flow cytometry has revealed that the distribution of the surface lectin on hemocytes represents a heterogeneous expression on a population basis, but no discrete cell subpopulations can be identified.     

A comparison of the cholinesterases of an oyster (Crassostrea virginica) and a clam (Macrocallista nimbosa).

Cholinesterase activities in the hearts and ganglia of an oyster (Crassostrea virginica) and a venerid clam (Macrocallista nimbosa) were measured and compared. Tissue extracts were partially purified by ammonium sulfate fractionation followed by gel column chromatography. Enzymatic activity was assayed spectrophotometrically; substrates were acetyl-, butyryl-, and propionylthiocholine (ATC, BTC, PTC). Kinetic constants characterizing each enzyme were derived. At all substrate concentrations, the hydrolysis rates of both clam enzymes were in the order: BTC greater than PTC greater than ATC. With oyster enzymes the ranking was ATC greater than or equal to PTC greater BTC. The specific activities of oyster heart and ganglion enzymes were similar. In contrast, clam ganglion extracts were 75-100 times more active than clam heart extracts and, with any substrate, had greater activity than either oyster enzyme. All enzyme preparations proved to be homogeneous on the bases of constant substrate activity ratios in successive column fractions, and of intermediate velocities with mixed substrates. Six cholinesterase inhibitors were tested. The specific acetylcholinesterase antagonist, B.W. 62C47, WAS MUCH MORE EFFECTIVE AGAINST OYSTER ENZYMES, WHILE THE SPECIFIC ANTIBUTYRYLCHOLINESTERASE, ISO-OMPA, almost totally inhibited calm enzyme activity, but had little effect on oyster. Eserine was the most effective inhibitor of both enzymes. In conclusion, the enzymes in oyster tissues are acetylcholinesterases, while clam enzymes are butyrylcholinesterases. Nevertheless, clam ganglion esterase is sifficiently active to hydrolyze the physiological substrate, acetylcholine. These results explain the long-observed differences in isolated heart pharmacology between ostreid and venerid bivalves.     

Studies on the gill ciliation of the American oyster Crassostrea virginica (Gmelin)

Latero-frontal, para-latero-frontal, and frontal ciliary tracts on the gill filaments of Crassostrea virginica (Gmelin) were studied with light microscopy and scanning electron microscopy. Latero-frontal cirri are complex structures composed of varying numbers of paired cilia. The multiple pairs of cilia which constitute a single cirrus are closely appressed for a portion of their length; they then branch laterally from the central axis in a plume-like fashion. Latero-frontal cirri of adjacent gill filaments create a filtration sieve which should be capable of retaining particles smaller than 1 μm in diameter. Para-latero-frontal cilia are short, closely spaced cilia arranged as a staggered row along the frontal side of each tract of latero-frontal cirri. Latero-frontal cirri and para-latero-frontal cilia occur on ordinary, principal, and transitional gill filaments. Frontal ciliary tracts of ordinary filaments are divided into a central, ventrally directed coarse tract, flanked on either side by a dorsally directed fine ciliary tract. The coarse tract is covered by cirri which are comprised of five to eight cilia, while the fine frontal tracts are made up of individually functioning cilia. The frontal ciliary tracts of principal and transitional filaments bear only dorsally directed fine cilia. The unique direction of effective beat of the coarse frontal cirri of ordinary filaments, in combination with the action of fine frontal cilia and the strategic location of mucus producing cells, is used to describe a possible mechanism for the sorting of filtered particles.     

Use of the 16S-23S rDNA internal transcribed spacer of Roseovarius crassostreae for epizootiological studies of juvenile oyster disease (JOD)

Juvenile oyster disease (JOD) in Crassostrea virginica is caused by the marine bacterium Roseovarius crassostreae. Although the 16S rRNA genes of the bacterial isolates exhibit little variation, 2 genetic signatures (GSI and GSII) may be discerned by Ava I digestion of the 16S-23S internal transcribed spacer (ITS). In this study we analyzed isolates from JOD epizootics throughout the northeastern USA (including affected adults for the first time) to better understand how oyster populations encounter and become affected by the pathogen. Isolates from a given epizootic usually had the same ITS signature; however, the involvement of both genetic signatures was occasionally detected, even within the same oyster. Sequencing was used to localize the variable Ava I site to a 100 bp region of low sequence identity, and detection of additional base changes resulted in the identification of 11 distinct genotypes. One genotype was found only in Martha's Vineyard, Massachusetts, USA and persisted in JOD survivors. Two genotypes were associated with Maine epizootics, and both were believed to be unique to that region until 2004, when one was detected in Martha's Vineyard among oysters that had survived colonization by the local genotype. Apparent competition between those 2 genotypes was also detected among a population of juveniles. Five genotypes were found only in New York, and the other 3 were isolated from both New York and from around Cape Cod, Massachusetts. Relationships between the geographic occurrence and phylogenetic relatedness of genotypes were compared with regional current patterns to identify possible mechanisms controlling their distribution.     

Occurrence of symbiotic turbellarians in the oyster,Crassostrea virginica

Crassostrea virginica was collected from several locations where it is cultured, both along the Northumberland Strait of New Brunswick and Malpeque Bay on the coast of Prince Edward Island. The oysters were found with two turbellarians on their gills. Urastoma cyprinae (Graff) was found in the oysters mostly during the warmer months of the year in numbers averaging as high as 50 worms per host (N = 50) and with as much as 78% of the host population infected (N = 100). Paravortex gemellipara (Linton) was also found during warmer months, but much less frequently or abundantly.Both male and female oysters were found to have U. cyprinae. No eggs or recent young of U. cyprinae were found in hosts; female-mature individuals of P. gemellipara with young were found from June through August.     

Modeling studies of the effect of climate variability on MSX disease in eastern oyster (Crassostrea virginica) populations

Eastern oysters (Crassostrea virginica) often undergo epizootics of MSX (Multinucleated Spore Unknown) disease, which is caused by the protozoan pathogen, Haplosporidium nelsoni. The disease has been present in oyster populations in the mid-Atlantic United States since the 1950s. During the 1980s and 1990s, it became established further north along the east coast of the United States. To investigate the factors underlying the northward progression of MSX disease, a model that simulates the host–parasite–environmental interactions was used. The model is physiologically-based and is structured around the transmission, proliferation and death rates of the parasite. Environmental conditions of temperature, salinity and oyster food supply provide the external forcing that results in variations in the biological rates. For this study, environmental data sets, both average and extreme, were obtained at a site in upper Chesapeake Bay for 1986 through 1995. This site is in the middle of one of the most productive oyster growing regions on the east coast of the United States; thus, both short- and long-term changes in the environment measured here realistically reflect conditions experienced by important oyster populations. The effect of short-term high-salinity (drought) or low-salinity (wet) conditions on MSX disease prevalence and intensity was relatively small because the average salinity regime already favors maximum parasite activity at this site. Even the extreme low salinity events were not low enough to significantly inhibit the parasite. Similarly, simulations using short-term high-temperature extremes for the same site showed only minor deviations from the average pattern because average temperatures were already high enough to support parasite development. In contrast, the measured low temperature conditions, applied for a single year, caused a dramatic reduction in parasite activity, which extended over a 2-year period. Additional simulations show that overall food supply to the host is of little consequence in determining the basic disease pattern; however, the timing of maximum food supply provided to the host, relative to specific times in the parasite life cycle, is important in determining whether or not the parasite attempts sporulation or undergoes density-independent growth. Simulations that test a sequence of changing environmental conditions show that when a year with cold winter temperatures (less than 3 °C) is followed by a year of low salinity (less than 15 ppt), prevalences and intensities of MSX disease are greatly reduced, with the disease becoming almost absent in the oyster populations; however, the disease returns when average environmental conditions return. Simulations using progressive cooling or warming conditions indicate that winter temperatures consistently lower than 3 °C limit the long-term development of MSX disease. These simulations support the suggestion that climate warming is a contributing factor to the northward spread of MSX disease.     

Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica)

Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.     

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.     

Interindividual variation of malate dehydrogenase activity in the oyster Crassostrea virginica

Intertidal environments are dynamic, stressful niches and variation in physiological parameters may determine distribution and survival of individuals in a population. We demonstrated that mitochondria of the oyster Crassostrea virginica oxidize malate more readily than other Krebs cycle intermediates and investigated the level of interindividual variability in oyster malate dehydrogenase (MDH) activity and total protein content in muscle tissues. Both MDH activity and total protein evidenced a high level of interindividual variation in heart and adductor among a sample of more than 50 oysters. Normalization to total DNA failed to explain the variation in either MDH activity or protein content of phasic adductor and explained less than 40% of the variation in heart. This range of MDH titers defines a continuum of biochemical phenotypes important for understanding the relative selection forces operative on metabolic pathways within the muscles of the oyster.     

Concentration of Escherichia coli in sediments as an indicator of the sanitary status of oyster (Crassostrea virginica) aquaculture sites

The objective of the study was to determine whether there are differences in Escherichia coli counts in relation to seasonal and/or spatial distribution patterns in a conditional shellfish‐growing zone located in the Richibucto estuary, New Brunswick, Canada. E. coli concentrations in surface water, sediments, suspended and bottom cultured American oysters (Crassostrea virginica) were determined. Contamination levels in water were significantly lower than in sediments, bottom and suspended cultured oysters (P < 0.01; P < 0.05, P < 0.01, n = 303, respectively). No significant difference in fecal contamination was observed between suspended and bottom cultured oysters (P > 0.05, n = 303). Seasonal variations (temporal) had a significant influence on fecal coliform contamination of all components (P < 0.01, n = 303, linear model). E. coli concentration levels were as much as ten times higher in sediments than in water. Furthermore, results suggest that E. coli concentration levels in sediments are a more reliable indicator of the sanitary status of a grow‐out operation than E. coli levels in water. Considering only sampling dates when contamination levels in oysters exceeded the established threshold of 230 MPN, less than 50% of the cases were predicted by water contamination while as many as 89.9% of the samples were predicted by sediment contamination using the proposed threshold of 75 MPN per 100 g. This paper emphasizes the importance of acknowledging the significant role sediments play in the dynamics of contamination by E. coli in areas that are currently exploited or are being considered for shellfish aquaculture. The presence of E. coli in sediments should not be overlooked as an integral factor in assessing the environmental sanitary status.