Cyrille Francois

Ostreid herpesvirus 1 detection and relationship with Crassostrea gigas spat mortality in France between 1998 and 2006

Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.

Can the protozoan parasite Bonamia ostreae infect larvae of flat oysters Ostrea edulis

Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It can be detected in juveniles but mortalities mainly affect oysters which are more than 2 years old. The parasite is usually observed inside haemocytes and sometimes free, notably in gill epithelia suggesting a parasite release through this organ. However, the infective form and ways of entry and release remain undetermined. Flat oysters incubate their larvae in their pallial cavity for 8-10 days before releasing them into the water column. Flat oysters in Bay of Quiberon in South Brittany (France) are known to be infected with B. ostreae since 1979 and is the most important area in France for O. edulis spat collection. Flat oysters incubating larvae were sampled in this area during summertime between 2007 and 2009. Both adults and larvae were preserved and assayed by PCR and in situ hybridisation (ISH). PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific labelling could be detected by ISH in gills, digestive system, gonad and mantle in adults and in the epithelium surrounding the visceral cavity of some larvae. Our results demonstrate that larvae can be infected with B. ostreae. Larvae might thus contribute to the spread of the parasite during their planktonic life. In addition, their transfer for aquaculture purpose should be controlled especially when they are exported from infected zones.

One Perkinsus species may hide another: characterization of Perkinsus species present in clam production areas of France

Although clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis.

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host-parasite relationship.