Oyster Strains Research Articles

Molecular analysis of an oyster-related norovirus outbreak

Background Contaminated raw oysters were implicated in a severe outbreak of norovirus (NoV) gastroenteritis affecting 30 restaurant guests. Objectives To define the outbreak source by using molecular methods to characterize NoV strains detected in patient and oyster samples. Study design Molecular epidemiological studies based on nucleotide sequencing and phylogenetic analyses of patient and oyster NoV strains, and comparison to background dataset. Results NoV genotype (G) I.1 was detected in the one patient stool analyzed by in-house TaqMan real time RT-PCR and classical nested RT-PCR targeting NoV RNA-dependent polymerase (RdRp, 285 nt), and by nested RT-PCR targeting RdRp-capsid-poly(A)-3′ (3085 nt). Patient strain showed ≥99% similarity (285 nt) with three NoV strains detected in two of five oysters examined by classical nested RT-PCR (RdRp). A third oyster tested positive for NoV GII.3. Phylogenetic analysis showed clustering of patient and oyster strains related to this outbreak with GI.1 strains from previous local outbreaks, and mussel studies. Conclusions Sequence data revealed ≥99% similarity (285 nt) between NoV GI.1 strains detected in patient stool and suspect oysters, linking the contaminated oysters to the outbreak. Identification of human NoV GI and GII strains in oysters indicated contamination of human fecal origin, presumably from inappropriate storage in the harbor. Comparative long-fragment analysis of the patient strain revealed 99% similarity (3085 nt) with NoV GI.1 strains detected in previous outbreaks and environmental mussel studies from West Sweden, 87% with M87661 (Norwalk68) and 96% with L23828 (SRSV-KY-89/89/J). These results indicated considerable genomic stability of NoV GI.1 strains over time.

Transcriptome profiling of selectively bred Pacific oyster Crassostrea gigas families that differ in tolerance of heat shock.

Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43 degrees C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion.

A genetic test for recruitment enhancement in Chesapeake Bay oysters, Crassostrea virginica, after population supplementation with a disease tolerant strain

Many of the methods currently employed to restore Chesapeake Bay populations of the eastern oyster, Crassostrea virginica, assume closed recruitment in certain sub-estuaries despite planktonic larval durations of 2–3 weeks. In addition, to combat parasitic disease, artificially selected disease tolerant oyster strains are being used for population supplementation. It has been impossible to fully evaluate these unconventional tactics because offspring from wild and selected broodstock are phenotypically indistinguishable. This study provides the first direct measurement of oyster recruitment enhancement by using genetic assignment tests to discriminate locally produced progeny of a selected oyster strain from progeny of wild parents. Artificially selected oysters (DEBY strain) were planted on a single reef in each of two Chesapeake Bay tributaries in 2002, but only in the Great Wicomico River (GWR) were they large enough to potentially reproduce the same year. Assignment tests based on eight microsatellite loci and mitochondrial DNA markers were applied to 1579 juvenile oysters collected throughout the GWR during the summer of 2002. Only one juvenile oyster was positively identified as an offspring of the 0.75 million DEBY oysters that were planted in the GWR, but 153 individuals (9.7%) had DEBY ×wild F1 multilocus genotypes. Because oyster recruitment was high across the region in 2002, the proportionately low enhancement measured in the GWR would not otherwise have been recognized. Possible causes for low enhancement success are discussed, each bearing on untested assumptions underlying the restoration methods, and all arguing for more intensive evaluation of each component of the restoration strategy.

Evaluation of resistance to Dermo in eastern oyster strains tested in Chesapeake Bay

We investigated growth, Dermo disease, and survival for nine groups of oysters, Crassostrea virginica (Gmelin) cultivated in Chesapeake Bay (CB). Five regional strains (upper CB, North Carolina (NC), South Carolina (SC), Louisiana (LA) and LA triploids) and four additional hybrid strains (CB oysters mated with NC, SC, LA and Texas (TX) oysters) were held in floating rafts at three locations representative of lower CB: ‘low’ salinity (3–14 g L−1), ‘moderate’ salinity (5–20 g L−1) and ‘high’ salinity (14–24 g L−1). At each site, patterns of growth and incidence of infection with Perkinsus marinus (Levine), the causative agent of Dermo disease, were similar. However, mortality trends were markedly different at each site; the CB strain being notable for accelerated mortality following infection with P. marinus. In addition, hybrids between CB and all four of the regional strains exhibited similar accelerated mortality in response to infection. Mortality was strongly correlated with infection only at the high salinity site implicating interaction of differences in both oyster strain and virulence of Dermo between moderate and high salinity areas as factors in differential mortality across sites.

Resistance of Dermo in eastern oysters, Crassostrea virginica (Gmelin), of North Carolina but not Chesapeake Bay Heritage

Growth, intensity of Perkinsus marinus (Levine) infection, and survival of synchronously spawned North Carolina (NC) and Chesapeake Bay-heritage (CB) oysters, Crassostrea virginica (Gmelin) were evaluated under standard tray culture conditions at several sites in both regions (Wye River, Maryland; Mobjack Bay, Virginia; Pamlico River, NC and Bogue Banks, NC). Infection prevalence reached 100% in oysters held at all high- and moderate-salinity sites, at which time the CB strain ceased to grow. Shortly after growth ceased, CB oysters exhibited mortality that rapidly progressed to 100%. Unlike the CB strain, growth continued in the NC strain despite high P. marinus prevalence. When mortality did occur in the NC strain, at a reduced rate of 37–40%, it was associated with higher intensity of P. marinus than the infection intensity correlated with death of CB oysters. At the low-salinity site in NC, P. marinus infection persisted at low weighted prevalence throughout the latter portion of the culture period but was not associated with mortality of either strain. These trends in growth and disease resistance for the two strains demonstrate that aquaculture performance is related to the level of disease resistance in oyster strains, salinity of water in growing areas and virulence of P. marinus.

Stappia alba sp. nov., isolated from Mediterranean oysters

Three bacterial strains isolated from oysters recovered at the Spanish Mediterranean coast have been phenotypically and genetically characterized. The results of the phylogenetic analysis based on almost complete 16S rDNA sequences clustered all three strains together with 99.9% average sequence similarity and situated them in the neighbourhood of the genera Stappia, Roseibium and Pannonibacter, Stappia aggregata being their closest neighbour with sequence similarities between 98.8% and 98.9%. DNA–DNA hybridization experiments using DNA of strains 5OM6 T and S. aggregata CECT 4269 T as reference DNAs confirmed the independent status at species level of the oyster isolates. Phenotypically, they can be distinguished from the closest relatives by the ionic requirements, growth temperatures and use of carbon compounds. We propose these oyster strains constitute a new species of Stappia, for which the name Stappia alba sp. nov. has been chosen, and strain 5OM6 T (=CECT 5095 T=CIP 108402 T) as its type strain.

Perkinsosis in molluscs: A review

The genus Perkinsus includes protistan parasites infecting marine molluscs throughout the world, some of which are associated with mass mortalities. Life cycle involves vegetative proliferation within the host, by which a cell named trophozoite undergoes successive bipartitioning. Other stages have been observed in vitro or in vivo, depending on the species: hypnospore, zoosporangium and zoospore. Molecular taxonomy supports a close affinity be- tween dinoflagellates and Perkinsus spp. Six species of Perkinsus are currently considered valid: P. marinus, P. olseni, P. qugwadi, P. chesapeaki, P. andrewsi and P. mediterraneus. Histology and, above all, incubation of host tissues in Ray's fluid thioglycollate medium (RFTM) are classic diagnostic methods. In addition, more sensitive and quicker molecular diagnostic techniques based on either immunoassays or PCR have been developed for Perkinsus spp. Epi- zootiological studies have shown a marked influence of water temperature and salinity on P. marinus infection in oys- ters Crassostrea virginica, thus determining parasite geographical range and temporal disease dynamics (seasonality). In vitro cultures have been established for four Perkinsus spp. Immune response to infection varies depending on host and involves phagocytosis or encapsulation of the parasite cells by host haemocytes. A polypeptide is secreted by clam Tapes philippinarum haemocytes that could kill the parasite. In vitro cultured P. marinus cells secrete proteases that are likely involved in degradation of host tissues. P. marinus can suppress the toxic oxygen radicals produced by host haemocytes. In addition to host death, sublethal effects caused by Perkinsus spp. (reduction of fecundity, growth, and condition) may have significant ecological and economic implications. Various strategies have been assayed to mitigate the consequences of P. marinus epizootics on the oyster industry: modifications of management/culture procedures, selective breeding to obtain resistant oyster strains, and the use of triploid oysters and allochthonous oyster species. Some chemotherapeutants have been proved to inhibit or kill parasite cells in vitro.

Dual disease resistance in a selectively bred eastern oyster, Crassostrea virginica, strain tested in Chesapeake Bay

Selective breeding efforts have yielded oyster strains, Crassostrea virginica, with improved survival and resistance against Haplosporidium nelsoni (MSX); however, because of susceptibility to the oyster pathogen Perkinsus marinus (Dermo), their utility has been limited in areas where the two parasites co-occur. Dual resistance to H. nelsoni and P. marinus was achieved through four generations of artificial selection of wild Delaware Bay oyster progeny at a site in the lower York River, Virginia, USA where both diseases are enzootic. During 1993–1995, survival, growth, and disease susceptibility of third generation Delaware Bay (F3-DEBY) oysters were evaluated at the York River site in comparison to that of similarly selected third generation James River, Virginia oysters (F3-JR), and first generation Louisiana oysters (F1-LA), whose parents were naturally selected in the wild for resistance to P. marinus. During 1997–1999, the performance of F4-DEBY was evaluated at three sites in Virginia in comparison to two groups of first generation oysters whose parents originated from Mobjack Bay and Tangier Sound, Virginia where both P. marinus and H. nelsoni are enzootic. In the presence of high infection pressure from both H. nelsoni and P. marinus, the F3-DEBY stock showed significantly higher survival and growth than either the F3-JR or F1-LA strain. After 15 months of deployment, 79% of F3-DEBY, 11% of F3-JR and 17% of F1-LA oysters were market size (≥76.2 mm) and cumulative mortality was only 16% in F3-DEBY as compared to 42% in F3-JR and F1-LA. At the termination of the study, F3-DEBY oysters exhibited 22% lower mortality than the F1-LA stock, which outperformed the F3-JR stock. Relative performance in respect to disease varied considerably with sample date; however, average H. nelsoni weighted prevalence varied such that F3-DEBY<F1-LA=F3-JR and average P. marinus weighted prevalence varied such that F1-LA<F3-DEBY<F3-JR. In the 1997–1999 trial, F4-DEBY oysters experienced 34–61% lower mortality, greater growth rate, and consistently lower prevalence and intensity of P. marinus than either Mobjack Bay or Tangier Sound oysters. H. nelsoni prevalences were very low (<12%) in all three stocks. This is the first study to demonstrate that reduced susceptibility to both P. marinus and H. nelsoni can be achieved through selective breeding.

Growth patterns in oysters, Crassostrea virginica , from different estuaries

We analyzed a data set collected over 15 yr, containing growth data from strains of eastern oysters, Crassostrea virginica (Gmelin, 1791), initiated from parent populations in Long Island Sound, Delaware Bay, and lower Chesapeake Bay. The long-term growth data proved to be a powerful tool for examining patterns of growth differentiation among separated populations of C. virginica. The oyster strains had been grown in a common environment in lower Delaware Bay for up to seven generations. We found that the oyster strains with origins in Long Island Sound were significantly larger over several generations than oyster strains from Delaware Bay and Chesapeake Bay. Chesapeake Bay oyster strains were larger than Delaware Bay oyster strains at 1.5 yr old, but Delaware Bay oysters were larger at 2.5 yr. Year-to-year variation in environmental conditions had a strong significant effect on absolute oyster size and the relative sizes of the oyster strains. Persistent differences between oyster strains from different origins over several generations support a hypothesis that these estuarine populations have experienced long-term genetically-based population differentiation. This result is consistent with hypotheses of population differentiation of oysters based on observations of local reproductive timing.

Latitudinal compensation in oyster ciliary activity

1. Theories of latitudinal compensation predict that individuals living in colder temperature regimes should physiologically compensate for the slowing of standard physiological rates, owing to the relatively low temperature of their local environments, by increasing their metabolic rate in colder water temperatures relative to individuals living in warmer water temperature regimes. 2. This hypothesis was tested with oyster strains originally from geographically separated populations that were raised in a common environment for seven generations. The physiological parameter measured was ciliary activity across a temperature gradient. 3. Support for the latitudinal compensation hypothesis was found: the strain originally from the colder temperature regime had more active cilia at lower experimental temperatures than individuals originally from the warmer temperature regime. Ciliary activity of the more northern Long Island Sound oysters was significantly greater than activity in the more southern Delaware Bay oysters at temperatures of –1, 2 and 6 °C. 4. These results suggest that there is genetically based physiological differentiation between these populations of oysters consistent with the latitudinal compensation for local temperature regime.

Overdominance in Early Life Stages of an American Oyster Strain

Overdominance in Early Life Stages of an American Oyster Strain

Acid-production and other Characters of Bacillus-Coli-Like Bacteria from Feces and Sewage

The ability to decompose carbohydrates with the formation of acid has long been recognized as one of the characteristics of Bacilluscoli-like bacteria. This property of acid-production is the basis for the isolation of B. coli on the Wurtz litmus-lactose-agar plate, and also for the separation of B. coli from its relatives, B. typhosus and B. paratyphosus, on the Conradi-Drigalski agar medium. The ability to ferment various substances has been further utilized as a basis for the subdivision of the colon-aerogenes group. In these studies on classification, however, attention has been focused upon gas-formation rather than upon acid-production. Browne,1 in an extensive study of certain factors influencing acidproduction, points out that Bacillus-coli-like bacteria isolated from oysters formed less acid from carbohydrates than those isolated from human stools, and he attributed the difference to a loss of fermenting power by the organisms in their passage through sewage from the intestines to the oysters. Unfortunately, this investigator did not differentiate the different types of organisms with which he was working. It is entirely probable that the smaller amount of acid observed among the oyster strains was due to a greater incidence of some particular type or species of Bacillus-coli-like microorganism rather than to a loss of fermenting power on the part of the intestinal forms. [After the completion of the experimental work for this paper, an article appeared by Clark and Lubs,2 who pointed out that bovine fecal strains of B. coli give rise to a higher H-f--ion concentration in glucose than do nonfecal (grain) strains.] The present investigation was undertaken to determine the following : 1. Do Bacillus-coli-like organisms from different sources (particularly animal sources) give rise to different amounts of acid in the