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Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman (R) PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. First part of this work consisted in assessing analytical sensitivity and specificity of the new PCR assay. Then, diagnostic performances were assessed by testing a panel of field samples with the new real-time PCR and currently recommended conventional PCR methods for the detection of M. refringens and Bonamia sp. Samples were collected from the main flat oyster production sites in France (N = 386 for M. refringens and N = 349 for B. ostreae). In the absence of gold standard, diagnostic sensitivity and specificity of the new PCR were estimated through Bayesian latent class analysis (DSe 87,2% and DSp 98,4% for the detection M. refringens, DSe 77,5% and DSp 98,4% for the detection of Bonamia sp.). Those results suggest equivalent performances for the detection of Bonamia sp. and an improved sensitivity for the detection of M. refringens compared to commonly used conventional protocols. Finally, the new PCR was evaluated in the context of an inter-laboratory comparison study including 17 European laboratories. Results revealed a very good reproducibility with a global accordance (intralaboratory precision) >96% and a global concordance (inter-laboratory precision) >93% for both targets, demonstrating that this new tool is easily transferable to different laboratory settings. This is the first assay designed to detect both Marteilia refringens and Bonamia sp. in a single step and it should allow reducing the number of analysis to monitor both diseases, and where relevant to demonstrate freedom from infection.

In the context of mass mortality outbreaks affecting adult and juvenile mussels since 2014 in France, two investigations were conducted to study mussel mortality under laboratory conditions in 2015. The first investigated the horizontal transmission of a putative causal agent of the mortality among two stocks of wild Mytilus edulis and the potential genetic resistance of adults and their progenies. The second evaluated the resistance of pure and hybrid crosses of M. edulis and M. galloprovincialis at the spat stage to a mortality outbreak. For the first time in France, we described several mortality outbreaks affecting spat and adult mussels under laboratory conditions. Mortality was observed for seawater temperatures ranging from 10 to 23 °C, although the significant mortality outbreaks occurred between 12 and 17 °C in adults and at 22 °C in spat. The disease screening revealed that no notifiable pathogen agent, as defined by OIE, was detected in the analyzed moribund mussels. Although bacteria belonging to the Splendidus clade were detected at high prevalence, with the exception of a mortality outbreak occurring in August, their role could not be defined in this study. Survivors of a mortality event occurring in the field in 2014 (Yves stock) still exhibited around 30% of mortality in 2015 under laboratory conditions, which indicated that they were either not fully resistant or that susceptibility varies with mussel age/size. This finding also suggests that a part of the survivors (or all) were carriers of a putative causal agent of the mortality. We demonstrated the horizontal transmission of a putative causal agent from the Yves stock to another stock of wild adult mussels (stock Agnas) which experienced 46% of mortality. In contrast, the Agnas stock had low mortality (5%) in the separate condition. Horizontal transmission of a putative causal agent from the wild adults to their hatchery-produced progenies was also observed. The progenies of the Yves stock, which survived a mortality outbreak in 2014, had lower mortality (27%) than those of the unselected Agnas stock (68%). Similarly, low mortality (

Although vibrios are frequently associated with marine organisms mortality outbreaks, knowledge on their ecology and pathogenicity is sparse, thus limiting disease management and prophylactic strategies. Here, we investigated V. aestuarianus infection onset and progression in the wild, taking advantage of a 'claire' pond: a semi-closed system with limited seawater renewal, theoretically more adapted to disease transmission. We showed a positive association of the bacteria with oysters, which can constitute a reservoir for the bacteria in the winter. Moreover, passage through oysters was found to be necessary for experimental disease reproduction as vibrios shedding from diseased oysters have higher infectivity than from in vitro grown. We next developed an experimental 'ecologically realistic' infection model in a mesocosm, allowing infection by natural route. By means of this non-invasive protocol, we analysed the pathogenesis of the bacteria and demonstrated the importance of haemolymph for initial colonization and the septicaemic nature of this disease.

Oyster species suffer from numerous disease outbreaks, often causing high mortality. Because the environment cannot be controlled, genetic improvement for disease resistance to pathogens is an attractive option to reduce their impact on oyster production. We review the literature on selective breeding programs for disease resistance in oyster species, and the impact of triploidy on such resistance. Significant response to selection to improve disease resistance was observed in all studies after two to four generations of selection for Haplosporidium nelsoni and Roseovarius crassostrea in Crassostrea virginica, OsHV-1 in Crassostrea gigas, and Martelia sydneyi in Saccostrea glomerata. Clearly, resistance in these cases was heritable, but most of the studies failed to provide estimates for heritability or genetic correlations with other traits, e.g., between resistance to one disease and another. Generally, it seems breeding for higher resistance to one disease does not confer higher resistance or susceptibility to another disease. For disease resistance in triploid oysters, several studies showed that triploidy confers neither advantage nor disadvantage in survival, e.g., OsHV-1 resistance in C. gigas. Other studies showed higher disease resistance of triploids over diploid as observed in C. virginica and S. glomerata. One indirect mechanism for triploids to avoid disease was to grow faster, thus limiting the span of time when oysters might be exposed to disease.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality outbreaks in European flat oyster stocks in Europe. As eradication and treatment are not possible, the control of the disease mainly relies on transfer restriction. Moreover, selection has been applied to produce resistant flat oyster families, which present better survival and lower prevalence than non-selected oysters. In order to better understand the mechanisms involved in resistance to bonamiosis, cellular and molecular responses of 2 oyster groups (selected oysters and wild-type oysters) were analyzed in the context of experimental injection and cohabitation infections. Cellular responses including non-specific esterases detection, ROS production and phagocytosis activity were analyzed by flow cytometry. Four genes homologous to those shown to be involved in immunity were selected (Inhibitor of apotosis OeIAP, Fas ligand OeFas-ligand, Oe-SOD, and OeEc-SOD) and monitored by quantitative reverse-transcription PCR (qRT-PCR). Infected oysters showed higher phagocytosis activity than controls. Infected selected oyster show a lower phagocytosis activity which might be a protection against the parasite infection. The expression of OeIAP and OeFas-ligand gene was significantly increased in selected oysters at 5 days post-injection. OeIAP gene expression appeared to be significantly increased in wild-type oysters at 8 days post-injection. Our results suggest that resistance to bonamiosis partly relies on the ability of the oysters to modulate apoptosis.

SUMMARYThe paramyxean parasite Marteilia refringens infects several bivalve species including European flat oysters Ostrea edulis and Mediterranean mussels Mytilus galloprovincialis. Sequence polymorphism allowed definition of three parasite types ‘M’, ‘O’ and ‘C’ preferably detected in oysters, mussels and cockles respectively. Transmission of the infection from infected bivalves to copepods Paracartia grani could be experimentally achieved but assays from copepods to bivalves failed. In order to contribute to the elucidation of the M. refringens life cycle, the dynamics of the infection was investigated in O. edulis, M. galloprovincialis and zooplankton over one year in Diana lagoon, Corsica (France). Flat oysters appeared non-infected while mussels were infected part of the year, showing highest prevalence in summertime. The parasite was detected by PCR in zooplankton particularly after the peak of prevalence in mussels. Several zooplanktonic groups including copepods, Cladocera, Appendicularia, Chaetognatha and Polychaeta appeared PCR positive. However, only the copepod species Paracartia latisetosa showed positive signal by in situ hybridization. Small parasite cells were observed in gonadal tissues of female copepods demonstrating for the first time that a copepod species other than P. grani can be infected with M. refringens. Molecular characterization of the parasite infecting mussels and zooplankton allowed the distinguishing of three Marteilia types in the lagoon.

Diploid and triploid Eastern oysters, Crassostrea virginica, were tested at 3 sites characterized by low or moderate salinity regimes in the Virginia part of the Chesapeake Bay from November 2005 through October 2007. Both diploid and triploid cultures were replicated 3 times by producing separate spawns from different broodstock. Ploidy had a generally consistent effect on the performance of C. virginica at the 3 test sites. At the end of the study, in October 2007, and across all sites, triploid oysters had lower cumulative mortality than diploids (-34%), and greater shell height (+25%), whole weight (+88%), and yield (+152%). as well as a higher proportion of market-size oysters (+114%) than diploids. Both diploids and triploids were similarly infected by Perkinsus marinus and, to a lesser extent, by Haplosporidium nelsoni. In a closer look, growth parameters (shell height growth, whole weight, yield, and percentage of marketable oysters) were always higher in triploids than in diploids regard...

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.

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

SUMMARYAlthough 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.

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.

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.

A study was conducted to investigate the involvement of bacteria in oyster mortalities during summer. Moribund and apparently healthy oysters were sampled during mortality events along the French coast and in rearing facilities, usually when temperature reached 19 degrees C or higher, and oysters were in the gonadal maturation phase. Hemolymph samples were aseptically withdrawn and submitted to bacteriological analysis. In healthy oysters, bacteria colonized hernolymph at low concentrations depending on the location. In most moribund oysters, bacteria were present in hemolymph and other tissues. These bacterial populations were more often diverse in oysters originating from the open sea than from facilities where animals were generally infected by a single type of bacterium. Only the dominant colonies were identified by phenotypic and genotypic characters (RFLP of GyrB gene and partial sequence of 16S rRNA gene). They belonged to a limited number of species including Vibrio aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus, and Pseudoalteromonas sp. The most frequently encountered species was V. aestuarlanus (56% of isolates), which was composed of several strains closely related by their 16S rRNA gene but diverse by their phenotypic characters. They appeared intimately linked to oysters. The species within the V. splendidus group were less prevalent (25% of isolates) and more taxonomically dispersed. A majority of the dominant strains of V. aestuarianus and V. splendidus group injected to oysters induced mortality, whereas others belonging to the same species, particularly those found in mixture, appeared innocuous.

Steinhausia mytilovuln is a globally distributed microsporidian parasite which infects the oocytes of the blue mussels Mytilus edulis and M. galloprovincialis. Despite the intensive monitoring effort made on mussel populations, the parasite has not previously been reported in France. We report herein on the occurrence of S. mytilovum in Mytilus sp. from 1 cultured and 2 natural populations on the northern coast of France, thus extending the parasite's known distribution northwards. We also report on the observation in 1989 of S. mytilovum in M, galloprovincialis from the Golfe de Fos area in the Mediterranean Sea (South of France), S. mytilovum was observed in the European hybrid zone between M. edulis and M. galloprovincialis, which therefore renders the exact taxonomic status of the infected hosts unknown, The prevalence of the parasite was low, which suggests that its effect on mussel populations was probably limited.

We report the occurrence of the microsporidian parasite Steinhausia sp. in the oocytes of the common cockle Cerastoderma edule in a natural population in France, where high mortalities occurred. Steinhausia sp. appeared primarily as sporocysts containing many small spores, and putative earlier developmental stages were also observed. Both its prevalence and infection intensity were low, and no host defence reaction was recognized, suggesting that Steinhausia sp. had no detrimental effect on C. edule. Its prevalence was higher in cockles lying on the sediment surface, but the significance of this observation could not be explained given the poor knowledge of the Steinhausia life cycle. The present data did not allow specific identification of the parasite, and further studies are required to determine whether Steinhausia sp. in the cockle is a new species, or a microsporidian infecting multiple host species.

International audience; Experimental infections and lethal dose 50% (LD$_{50}$) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 °C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 °C, revival and subsequent in vivo passages in fish. Experiments were performed 1, 8 and 23 months after storing the bacteria. Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages. In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage. In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 °C were found significantly attenuated. Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD$_{50}$. It was concluded that virulent strains of F. psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 °C were reliable methods for long-term preservation of virulence.