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Abstracts
Session: The State of Oyster Disease
Juvenile Oyster Disease: Progress and Directions in Research
Presented By:
Katherine J. Boettcher, University of Maine, boettche@maine.edu
It has been almost fifteen years since juvenile oyster disease emerged as a serious problem in the Northeast (especially in Maine and New York). Annual epizootics affect first-year seed during the nursery growout phase of commercial culture, and may kill more than 90% of a crop within a period of only a few weeks. Typically, signs such as uneven valve margins, proteinaceous shell deposits (conchiolin), and general emaciation immediately precede the onset of mortalities. No other species besides the Eastern oyster (Crassostrea virginica) is affected, and smaller animals (less than 25 mm shell height) are most vulnerable.
The etiological agent is believed to be a species of marine alpha-proteobacterium, which on the basis of phenotypic and phylogenetic analyses, represents a new taxon within the Roseobacter clade. This bacterium (proposed designation Roseimarina crassostreae, gen. nov, sp. nov.) is present as an almost pure culture on the tissue surfaces of affected animals, and has been associated with all documented epizootics in Maine since 1997. Last year, isolates were also recovered from two groups of animals that were survivors of JOD-epizootics in New York. Mortalities have been induced by experimental exposure to the bacterium, and the presence of Roseimarina in affected animals was confirmed by microbiological analyses. Experiments with laboratory held animals indicate that colonization by Roseimarina interferes with the ability of the animal to filter-feed, and that death results from some combination of starvation and/or bacterial toxin.
Low (< 5% cumulative) mortalities were observed in a population of fairly large animals (greater than 25 mm) in 2002. Still, conchiolin deposits were observed in 9% of oysters, and these animals were found to be extensively colonized by Roseimarina. The soft body tissues in oysters with conchiolin were still in good to fair condition, thus, JOD did not seem to be progressing beyond the initial stages. This was the first extensive sampling of (and subsequent recovery of Roseimarina from) larger animals with JOD signs but in the absence of significant mortalities.
The range of JOD is expanding, but for largely unknown reasons. (Some introductions are, however, clearly the result of transfer of infected seed). Just recently, the discovery of small variations in a DNA region between two ribosomal genes (the internal transcribed spacer, or ITS) has provided the basis for the first epidemiological studies of JOD. Restriction-digest analyses of PCR-amplification products revealed two genotypes: the first (GT1) was associated with JOD in Maine in 1997 and 1998, and a second (GT2) was recovered from all examined Maine epizootics in 2000, 2001, and 2002. Representatives of both genotypes were discovered among the 2002 New York isolates, and these regions are being sequenced to determine overall percent similarity to the Maine strains.
A PCR-diagnostic assay based on amplification of the ITS region is being developed in our lab. The specificity of the assay has been confirmed, and amplifications are successful with cells taken directly from culture plates. There are, however, still some problems with assay sensitivity using samples taken directly from animals (i.e. before recovery in culture). It appears that unknown factors in mucous or other extracellular products of oyster tissue are interfering with amplification. We are exploring several strategies for dealing with these contaminating substances.
Efforts are also directed toward the production of immunological reagents for detection of the pathogen. A polyclonal antibody has been produced in chickens from a 'cocktail' of Roseimarina cells (in various physiological states grown on solid and liquid media). In agglutination tests, the affinity-purified antibody shows strong reactivity to cells of Roseimarina, and no cross-reactivity with the most closely related bacterial species (Roseovarius tolerans). In tests of isolates obtained from 2002 epizootics in Maine and New York, a 100% correlation was observed between those isolates determined to be Roseimarina by the PCR-assay and those agglutinated by the antibody. Further, no agglutination has been observed with cells from colonies displaying morphologies similar to Roseimarina but which tested negative in the PCR-assay. Western blots will be performed to identify the specific proteins in Roseimarina which are recognized by the sera. Important applications include a slide-based indirect fluorescent antibody test (IFAT) and in situ detection procedures to study host-pathogen interactions.
Very little is known about the process by which Roseimarina colonize and persist on surfaces of oyster tissues. Despite being in near constant contact with the ambient seawater, the outer tissue surfaces of healthy oysters remain essentially free of any attached bacteria. Yet, clearly Roseimarina can circumvent host defenses and become established on such tissues. We are currently investigating the hypothesis that oyster hemocytes are not always able to: (i) recognize or, (ii) phagocytose and destroy Roseimarina colonists. (Because oysters lack an antibody-based immune system, these cells form the primary line of immunological defense). Assays that measure the degree of hemocyte immunocompetence are being performed in both microtiter plates (where a reduction of a dye correlates with cell viability), and with standard-plate count techniques. We hope to define those conditions which are optimal for C. virginica to defend itself against Roseimarina, and use such information for the development of targeted prevention and/or remedial measures.
Of course, growers are primarily in need of options to help manage the risk of JOD. Previous work at the University of Maine demonstrated the clear relationship between bigger oysters and higher survival rates. These results led to the practice of early deployment of oyster seed such that most reach the 'size refuge' by the time epizootics typically occur (late Summer to early Fall). Still, for growers who conduct operations on enzootic systems (such as Maine's Damariscotta River), production becomes limited to a short time in the best growing season. An alternative solution may be that of 'biocontrol'. In 1998, a bacterium was discovered to be abundant in oysters which were apparently immune to JOD. We hypothesized that this bacterium might be a useful probiotic, and an experiment was conducted last year (in cooperation with a commercial grower) to evaluate its effectiveness. The results are inconclusive because there was a minimal natural impact of JOD, but they are nonetheless encouraging. Specifically, although control oysters did not suffer high mortalities, they did exhibit signs of JOD (notably conchiolin deposition). Such signs were absent in animals treated with the potential probiotic.
In much of this we are largely dependent upon the cooperation of oyster growers (one reports losing 50% of his production each year to JOD). However, the true impact and range of JOD is really not known, due to the reluctance of growers to report outbreaks. We've also found that during years of minimal mortality, JOD may be misdiagnosed (e,g, dismissed as 'normal' losses). Thus, one challenge is teaching growers to recognize JOD - in addition to advising them of the risks of seed transfer. It is likely to be more difficult to convince culturists to be more forthcoming and report suspected cases of JOD. They must first be persuaded of the importance of an accurate understanding of the range and site-specific losses caused by this disease.
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