Rose-Marie Le Deuff

Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France.

A PCR-based procedure for detecting a herpes-like virus that infects the Japanese oyster, Crassostrea gigas, in France was developed. Two primers were designed to provide specific amplification products ranging in size from 917 to 1001 bp when carried out on oyster herpes-like virus DNA. No amplification was observed of oyster genomic DNA nor of the DNA from vertebrate herpesviruses. Crude samples were prepared and submitted to nested PCR, allowing amplification of DNA fragments of the expected size when carried out on infected larval and spat samples. The procedure used to prepare the sample for PCR was found to be critical because of the presence of unidentified substances in oyster tissues that inhibit the PCR reaction. A rapid and convenient sample preparation using ground tissues allowed a sensitive detection of the herpes-like virus infected oysters. The ability of the defined PCR protocol to diagnose herpes-like virus infections in oysters was compared to the transmission electron microscopy technique using 15 C. gigas larval batches with or without mortalities. PCR amplification is as sensitive a diagnostic assay for herpes-like virus as transmission electron microscopy. However, the nested PCR protocol is more convenient and less time consuming. The relationship between reported mortalities among C. gigas oyster spat and herpes-like virus DNA detection by PCR was also investigated. Statistical analysis showed that virus detection and mortalities are correlated. This observation highlights the importance of studying the causative role of herpes-like virus in oyster spat mortalities.

Isolation and culture of heart cells from the European flat oyster,Ostrea edulis

The present study reports a culture technique for heart tissues of the European flat oyster,Ostrea edulis. Heart tissues of flat oysters were dissociated by a trypsin-EDTA treatment and a mechanical action in a Dounce-type homogeneizer. Then, dissociated cells were cultured in three different synthetic media, in pure sea water or in sea water mixed with sterile filtered Japanese oyster,Crassostrea gigas, hemolymph. All these media were supplemented with 10% of fetal bovine serum. Cultures were grown at 20°C in previously Poly-D-Lysin coated flasks or culture wells. The optimized medium, 3% L15 medium (w/v) in sterile sea water mixed with Japanese oyster hemolymph (1:1) and supplemented with 10% of fetal bovine serum gave the best result. Morphological characterization for the cardiac cultured cells was performed. Thus, cell monolayers of dissociated heart tissues consisted essentially of hemocytes and large granular pigmented cells.

Primary culture of Pacific oyster,Crassostrea gigas, heart cells

Pacific oyster,Crassostrea gigas, is the most economically important specie to the world shellfish breeding. It is important to note that infectious diseases, particularly viruses, may be hazardous for theC. gigas live-stocks. The study of these viral diseases and the development of diagnosis method need the establishment of in vitro methods for viral multiplication. As no oyster cell line is available actually, we have developed a procedure for primary culture of heart cells which could enable to study molluscan viruses in vitro, and could also provide a diagnosis method based on the search of eventual cytopathogen viral effects. Cells fromC. gigas ventricle of heart were dissociated by trypsin-EDTA treatment and the mechanical action of a Dounce type homogeneizer. The cells were inoculated in previously poly-D-lysin coated flasks. The optimised culture medium was L-15 (Leibovitz) prepared three fold concentrated, then diluted half with sea water, this mixture was supplemented with 10% FCS and 5%C. gigas hemolymph. Different cell types could be identified by transmission electron microscopy analysis, as mostly cardiomyocytes, fibroblast-like cells and pigmented cells, but also haemocytes were present in the cultures.