First report of a Mikrocytos-like parasite in European oysters Ostrea edulis from Canada after transport and quarantine in France.

As part of a disease resistance experiment, 112 apparently healthy European flat oysters Ostrea edulis L. were exported from Canada (Nova Scotia) into France to test their susceptibility to Bonamia ostreae infection. Twelve oysters died in transit and 17 others died within 2 wk of laboratory quarantine acclimation. All oysters were examined histologically, and the 17 that died during quarantine were assayed for microcells (Bonamia sp. and Mikrocytos mackini) using molecular techniques. A microcell parasite was detected in the connective tissue of 5 of the 112 oysters. Morphological appearance, tissue affinity and molecular characterization through PCR, in situ hybridization (ISH), fluorescence in situ hybridization (FISH) and sequencing revealed a protist related to M. mackini. This is the first report of a parasite of the genus Mikrocytos in a species belonging to the genus Ostrea from the Atlantic Ocean.

Bonamia-like parasite found in the Suminoe oyster Crassostrea rivularis reared in France.

Considering the economic importance of the Pacific oyster Crassostrea gigas to the French shellfish industry, the appearance of major diseases in this species could cause dramatic decreases in production. Suminoe oysters, of the non-indigenous species Crassostrea rivularis (Gould), were introduced into France to test their ability to adapt to local conditions. These oysters were imported after careful examination, and were maintained in laboratory quarantine. Some mortalities occurred 7 mo after importation. Histological and electron microscope examinations of 9 dead specimens revealed a parasite presumed to be a Bonamia-like protozoan. This is the first report concerning a parasite of the genus Bonamia in a species belonging to the genus Crassostrea. Thus, C. rivularis is not considered to be a suitable substitute for C. gigas in France.

Concomitant herpes-like virus infections in hatchery-reared larvae and nursery-cultured spat Crassostrea gigas and Ostrea edulis.

Concomitant sporadic high mortalities were reported in France in May 1994 among batches of hatchery-reared larval Pacific oysters Crassostrea gigas and European flat oysters Ostrea edulis in 2 hatcheries, and in June and July 1994 among batches of cultured spat of both species in a shellfish nursery. Histological observation showed the presence of cellular abnormalities in moribund animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in infected larvae and spat of both oyster species. This is the first description of a herpes-like virus infection in larval O. edulis. Viruses observed in diseased larvae and spat of both species are similar with respect to ultrastructure and morphogenesis. They were detected simultaneously in C. gigas and O. edulis larvae and spat, indicating possible interspecific transmission. Moreover, these viruses are associated with high mortality rates in both oyster species. An electron microscopic examination revealed hemocytes with condensed chromatin and extensive perinuclear fragmentation of chromatin. These data suggest that herpes-like viruses infecting oysters may induce apoptosis in oyster hemocytes.

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast.

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations.

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.

Shell disease in eastern oysters, Crassostrea virginica, reared in France.

Progeny of eastern oyster, Crassostrea virginica, introduced into France in 1992, were reared in IFREMER facilities to test their growth performances. During the summer of 1993, sporadic mass mortalities (80-90%) occurred among C. virginica spat reared in the IFREMER laboratories in La Tremblade (Charente Maritime, France) and Bouin (Vendée, France). Affected oysters presented mantle retraction and deposition of an anomalous conchiolin layer on the inner surface of the shell. The incidence of oysters with gross signs exceeded 80%. No obvious pathogen was identified in soft tissues by histology and transmission electron microscopy (TEM). However, histological examination showed the presence of anomalous basophilic round structures, 0.5-1 microm in diameter, in gill and mantle connective tissues. These extracellular Feulgen-negative structures reacted positively with the von Kossa stain. TEM examination on mantle and gill samples in diseased spat showed that the basophilic bodies consisted of concentric deposits of an amorphous substance interpreted as containing calcium. These observations may indicate that the mineralization process in spat shells was disturbed without exact determination of the cause. Based on the similarities of the gross signs to those reported in juvenile eastern oysters in the United States, we believe that the cause of the mortalities observed in France was probably the Juvenile Oyster Disease. Moreover, we report for the first time the detection of anomalous amorphous structures in gill and mantle connective tissues associated with mortalities and deposition of an anomalous conchioloin layer on the inner shell surface in C. virginica spat.

Detection of Bonamia ostreae based on small subunit ribosomal probe.

Bonamia ostreae is a protozoan parasite of the flat oyster, Ostrea edulis, which has caused significant loss of oysters in Europe over the last decade. B. ostreae was purified from infected flat oysters and DNA was extracted. The nearly complete small subunit rDNA gene of B. ostreae was amplified using universal oligonucleotides and the PCR product was cloned and sequenced. BLAST research with this sequence revealed similarities to Haplosporidium nelsoni, Haplosporidium costale, and Minchinia teredinis. These data suggest that B. ostreae may be included in the genus Haplosporidium. Specific B. ostreae primers were designed for labeling, by PCR, a probe. This probe was successfully used by in situ hybridization to detect B. ostreae in infected fiat oysters, thus confirming the accuracy of this SSU rDNA sequence. The probe lead also to the detection of Bonamia sp. in infected Tiostrea chilensis and H. nelsoni in infected Crassostrea virginica but not Mikrocytos mackini infected Crassostrea gigas. These primers were also used to detect B. ostreae from infected oyster tissues by PCR. This B. ostreae SSU rDNA gene sequence provides genetic information as a first step toward elucidation of the taxonomic boundaries among the microcell organisms. Moreover, the development of DNA detection assays will be valuable specific diagnostic tools.