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Vibriosis is one of the most usual infection diseases in bivalve mollusks, particularly affecting seeds and larvae, which are more susceptible than adults to these bacterial infections. The devastating effect of vibriosis in shellfish hatcheries is aggravated by the increasing resistance of many Vibrio species to traditional antibiotics, highlighting the need to find new antimicrobial agents. Antimicrobial peptides (AMPs) are a promising alternative, however, their low stability can be a handicap for their practical application. In this study, the 3D structure and the stability of a synthetic all-D-amino acid peptide (D-Caerin) to proteases and extreme pH conditions have been analyzed and compared with its corresponding natural L-enantiomer peptide, Caerin 1.1. Moreover, the antimicrobial activity of D-Caerin has been tested in vitro against the control bacteria Micrococcus luteus CECT 245; and four Vibrio species: Vibrio aestuarianus CECT 625 T, Vibrio anguillarum CECT 522 T, Vibrio harveyi CECT 525 T and Vibrio tapetis CECT 4600 T, which are among the most representative causative agents of vibriosis in aquaculture. Our results demonstrate that D-Caerin contains two left-handed alpha helices, and is more stable and effective against the bacterial species tested than its corresponding natural L- counterpart. The bioactivity of D-Caerin has also been tested in vivo, in clam seeds infected with a mixed inoculum of these vibrio species. These preliminary assays show that D-Caerin-treated specimens exhibit higher survival rates after infection, demonstrating the superior stability of D-Caerin and its effectiveness against vibriosis-causative agents., This research was funded by the Andalusian government (I+D+i-JAPAIDI-Retos projects 2020-PY20_00728)

International audience; Vibrio aestuarianus infection in oyster populations causes massive mortality, resulting in losses for oyster farmers. Such dynamics result from host-pathogen interactions and contagion through water-borne transmission. To assess the spatiotemporal spread of V. aestuarianus infection and associated oyster mortality at a bay scale, we built a mathematical model informed by experimental infection data at 2 temperatures and spatially dependent marine connectivity of oyster farms. We applied the model to a real system and tested the importance of each factor using a number of modelling scenarios. Results suggest that introducing V. aestuarianus in a fully susceptible adult oyster population in the bay would lead to the mortality of all farmed oysters over 6 to 12 mo, depending on the location in which infection was initiated. The effect of temperature was captured by the basic reproduction number (R0), which was >1 at high seawater temperatures, as opposed to values

My PhD project aimed at investigating the molecular mechanisms at the basis of the interaction between Vibrio bacteria and shellfish in the bivalve models Crassotrea gigas and Mytilus galloprovincialis and to study the composition and dynamics of bivalve microbiota. Previous studies suggested that persistence of entrapped bacteria inside bivalve tissues depends, at least in part, on their capacity to survive to the hemolymph bactericidal activity, that is exerted by both hemocytes and serum soluble factors. In the first part of my PhD work, hemocytes of M. galloprovincialis were challenged with different pathogenic Vibrio strains (V. aestuarianus 01/032, V. aestuarianus 02/041, V. tasmaniensis LGP32, V. harveyi VH2, V. tapetis CECT 4600 and V. coralliilyticus ATCC BAA 450) in the presence or in the absence of the extrapallial protein present in M. galloprovincialis serum (MgEP), and of the whole hemolymph serum. In addition, C. gigas hemocytes were exposed to the bivalve pathogens V. aestuarianus 01/032 and V. aestuarianus 02/041 under the same conditions to better understand molecular basis of bacteria-hemolymph interactions in oysters. We observed that MgEP promotes D- mannose sensitive adhesion to and killing by hemocytes of the bivalve pathogens V. aestuarianus 01/032, V. aestuarianus 02/041, V. tasmaniensis LGP32 and V. coralliilyticus ATCC BAA 450. In addition, in the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 and V. aestuarianus 02/041 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of Vibrio strains to the antibacterial activity of oyster (susceptible to Vibrio infection) and mussel (resistant to Vibrio infection) haemolymph might partly depend on the fact that C. gigas serum lacks MgEP-like opsonins. These results may have important implications for improving bivalve depuration strategies and prevent diseases affecting bivalve production worldwide. In the second part of my thesis work, I studied the microbial communities associated to contrasting C. gigas samples collected during mortality episodes in different European sites. Real-time PCR targeting oyster pathogens (e.g. Ostreid herpesvirus 1 [OshV-1] and V. aestuarianus) and 16SrRNA gene-based microbial profiling were applied on a large number of C. gigas samples (n=525 and n=101 for qPCR and 16SrRNA gene profiling analysis, respectively) to extensively investigate the patterns and dynamics of oyster microbiota during mortality events. Comparative analysis of contrasting (e.g. infected vs not infected) C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. This represents to our knowledge, the largest study conducted so far to determine the composition and dynamics of farmed oyster microbiota.

Consumer preference for healthy and sustainable food products has been steadily increasing in recent years. Bivalve mollusks satisfy these characteristics and have captured ever-increasing market shares. However, the expansion of molluscan culture in worldwide and global trade have favored the spread of pathogens around the world. Combined with environmental changes and intensive production systems this has contributed to the occurrence of mass mortality episodes, thus posing a threat to the production of different species, including the Pacific oyster Crassotrea gigas. In the San Teodoro lagoon, one of the most devoted lagoons to extensive Pacific oyster aquaculture in Sardinia, a mortality outbreak was observed with an estimated 80% final loss of animal production. A study combining cultural, biomolecular and histopathological methods was conducted: (1) to investigate the presence of different Vibrio species and OsHV-1 in selected oyster tissues (digestive gland, gills, and mantle), (2) to quantify Vibrio aestuarianus and to evaluate the severity of hemocyte infiltration in infected tissues, (3) to produce post-amplification data and evaluating ToxR gene as a target for phylogenetic analyses. Results provide new insights into V. aestuarianus infection related to oyster mortality outbreaks and pave the way to the development of tools for oyster management.

International audience; Ostreid herpesvirus 1 (OsHV-1) and Vibrio aestuarianus are the two main pathogens affecting the production of French oyster (Crassostrea gigas). The absence of genetic correlation between the two diseases is promising for the development of stocks with dual resistance. Using unselected and selected oysters concerning enhanced resistance to OsHV-1 infection, we investigated the first generation of mass selection and the response to selection to increase the resistance to V. aestuarianus for two stocks. For each stock, four groups were produced in June 2013 using either parents unchallenged with the bacteria or counterparts that survived experimental infections by the bacteria. Thus, groups were unselected oysters for both pathogens, selected for either the virus or the bacteria, and dually selected for both pathogens. All groups of each stock were evaluated at the spat and juvenile sizes following experimental infection by V. aestuarianus in May 2014. Regardless of their level of selection for OsHV-1, oysters produced from parents that survived V. aestuarianus showed similar mortalities (47% and 53% for stocks A and B, respectively) during the bacterial challenge than those produced from unchallenged parents (43% and 56%, respectively). Thus, no positive response to selection at the first generation to increase the bacterial resistance was found at the spat and juvenile sizes. At the adult stage and with experimental infection with V. aestuarianus, only stock B showed a positive response to selection for increasing the bacterial resistance with a decrease in mortality of 14% in comparison with unselected oysters. Similar results were observed when oysters were tested for 27 months in the field with the absence of response to selection for stock A, while a 13% decrease in mortality was observed for stock B. For stock B, mortality at endpoint after 27 months in the field reached 89% for the control not selected at all, decreased to 84% for oysters selected for their resistance to the bacteria, was 53% for the oysters selected for their resistance to OsHV-1, and was 32% for oysters selected for dual resistance. Realized heritability estimated at the first generation of mass selection for stock B was higher for oysters selected for dual resistance, ranging from 0.47 to 0.80, than those only selected for V. aestuarianus, ranging from 0.05 to 0.30. Selection for dual resistance in C. gigas could limit the impact of both OsHV-1 and V. aestuarianus on oyster production

International audience; Summer mortality episodes in adult Pacific oysters have been described since the 1950s in various farming areas. Starting in 2012, a recrudescence of mortalities in commercial-sized oysters was first observed in France and then in Italy, with seasonality extension and translation later in the year. Moribund individuals collected during an event in Italy in December 2014 showed yellowish lesions of the mantle and adductor muscle. Histological examination revealed filamentous bacteria associated with necrotic areas. Quantitative PCRs targeting OsHV-1 and Vibrio aestuarianus detected only high loads of the pathogenic bacteria in tissues of symptomatic individuals. A lower diversity of the hemolymph microbiota was also evidenced in moribund individuals, with a predominance of Vibrio and Arcobacter species. A strain of Flavobacteriaceae was isolated from all the symptomatic individuals. Sequence analysis of the 16S rRNA gene identified the strain as Tenacibaculum soleae. When strain pathogenicity was tested by injection in adult individuals, it induced mortality rates of up to 45%, even in the absence of V. aestuarianus. As mortality occurred only 11 days post-infection, further investigation is needed to determine its effective virulence in natural conditions. This is the first description of a Tenacibaculum strain associated with bivalve mortalities.

Validation of a novel quantitative real-time PCR using TaqMan (R) minor groove binder (MGB) chemistry is described for sensitive and rapid detection of Vibrio aestuarianus, an increasingly important pathogen of Pacific cupped oyster Crassostrea gigas aquaculture. Primers and TaqMan (R) MGB hydrolysis probe were designed to specifically amplify a 58bp DNA fragment of the V. aestuarianus dnaJ gene. Real-time PCR selectivity was empirically tested using DNA extracted from isolates of V. aestuarianus and a selection of different aquatic bacterial species, including other Vibrio spp. Theoretical selectivity was assessed through sequence comparison using the NCBI BLAST similarity tool. Quantitative PCR plasmid standards were generated to test assay linearity, amplification efficiency and the limit of quantitation (LOQ), according to International Organisation for Standardisation ISO 16140 validation recommendations. LOQ ranged between 5 and 10 PCR copies, although the detection range extended beyond this with reduced precision. Applied performance was tested using C. gigas samples taken from a selection of Irish aquaculture sites. Increasing levels of V. aestuarianus, accompanied by the development of tissue pathology in examined oysters, were found at 1 site that was sampled repeatedly in 2013. Rapid, sensitive and reproducible detections of V. aestuarianus from C. gigas tissue samples were attained during this validation study with a small sample size, and a practical application for disease management is described.

Recent mass mortality outbreaks around the world in Pacific oysters, Crassostrea gigas, have seriously affected the aquaculture economy. Although the causes for these mortality outbreaks appear complex, infectious agents are involved. Two pathogens are associated with mass mortality outbreaks, the virus ostreid herpesvirus 1 (OsHV-1) and the bacterium Vibrio aestuarianus. Here we describe the interactions between these 2 pathogens and autophagy, a conserved intracellular pathway playing a key role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters.

Vibrio aestuarianus is an opportunistic environmental pathogen that has been associated with epidemics in cultured shrimp Penaeus vannamei. Hepatopancreas microsporidian (HPM) and monodon slow growth syndrome (MSGS) have been reported in cultured P. vannamei. In this study, we sequenced and assembled the whole genome of V. aestuarianus strain W‐40, a strain that was originally isolated from the intestines of an infected P. vannamei. The genome of V. aestuarianus strain W‐40 contains two circular chromosomes of 483,7307 bp with a 46.23% GC content. We identified 4,457 open reading frames (ORFs) that occupy 86.35% of the genome. Vibrio aestuarianus strain W‐40 consists primarily of the ATP‐binding cassette (ABC) transporter system and the phosphotransferase system (PTS). CagA is a metabolism system that includes bacterial extracellular solute‐binding protein. Glutathione reductase can purge superoxide radicals (O22−) and hydrogen peroxide (H2O2) damage in V. aestuarianus strain W‐40. The presence of two compete type I restriction‐modification systems was confirmed. A total of 42 insertion sequences (IS) elements and 16 IS elements were identified. Our results revealed a host of virulence factors that likely contribute to the pathogenicity of V. aestuarianus strain W‐40, including the virulence factor genes vacA, clpC, and bvgA, which are important for biofilm dispersion. Several bacitracin and tetracycline antibiotic resistance‐encoding genes and type VI secretion systems were also identified in the genome. The complete genome sequence will aid future studies of the pathogenesis of V. aestuarianus strain W‐40 and allow for new strategies to control disease to be developed.

Globally, Pacific oysters Crassostrea gigas are experiencing significant mortalities especially in warmer summer months, as a result of a complex aetiology and a strong association with pathogens such as ostreid herpes virus-1 microVar (OsHV-1 µvar) and bacteria Vibrio aestuarianus. Such mortalities threaten the future growth and sustainability of this industry worldwide. A better understanding of how these pathogens and diseases are interacting with their host and the environment at culture sites is needed, and new management and practical husbandry techniques are desired. This three-year study investigated the host-pathogen-environment interplay of Pacific oysters by carrying out field trials at the two largest culture sites in Ireland and by performing laboratory trials. The objectives of this study were to: (a) to understand how OsHV-1 µVar is becoming established at C. gigas culture sites. A five month field trial was undertaken in the spring and summer 2015. Green shore crabs Carcinus maenas and edible cockles Cerastoderma edule were collected at the oyster trestles and 500 m on the high shore at two culture sites, and were screened for OsHV-1 µVar by polymerase chain reaction (PCR), quantitative PCR (qPCR) and in situ hybridisation (ISH). OsHV-1 µVar was detected in both species by all diagnostics and was confirmed by direct sequencing. The mean prevalence of infection was 18.3% (n = 121/660) in Dungarvan and 16.3% (n = 155/952) in Carlingford Lough in C. maenas and 14.4% (n = 72/500) in Dungarvan and 13.6% (n = 121/890) in Carlingford Lough in C. edule. Following on from the field screening results, several 14 day laboratory trials were conducted to determine if naturally exposed crabs and cockles from culture sites could transmit OsHV-1 µvar to naïve oysters. OsHV-1 µvar was first detected in oysters cohabiting with exposed crabs on Day 4 and in oysters exposed to the cockles on Day 5. An overall prevalence of 6.5% in oysters held with crabs was observed while 4.4% was observed in the oysters held with cockles. Viral copies remained low (oysters exposed to crabs had an average of 1.0x 102 viral copies μl -1 of genomic DNA, oysters exposed to cockles had an average of 3.0 x103 DNA viral copies μl -1 of genomic DNA) in both trials, but this could be the result of trials being run at a low temperature (14 °C) less than the threshold temperature (16 °C) for replication of the virus. To the best of our knowledge C. maenas is the first non-bivalve species indicating infection of OsHV-1 µvar. (b) investigate the role of crabs, cockles and mussels as carriers or reservoirs for Vibrio spp.. In Ireland, Vibrio aestuarianus has been associated with C. gigas mortalities. V. aestuarianus was found at low infection intensities in C. maenas and C. edule and only at one of the main culture sites in 2015. Mussels Mytilus spp. were sampled from culture (2 hot spots) and non culture sites (

To enable the rapid and accurate identification of Vibrio splendidus-related and V. aestuarianus strains associated with Pacific cupped oyster Crassostrea gigas mortality, we developed a duplex Taqman real-time PCR assay and evaluated its efficacy. This technique proved to be rapid, sensitive, and specific and will be particularly valuable for epidemiologic studies.

L'eau qui alimente des structures d’élevage ou encore des plateformes expérimentales aquacoles (et potentiellement l’eau qui en est issue) peut être contaminée par des agents pathogènes infectant les coquillages, et plus particulièrement les huîtres, ou encore par des agents pathogènes entériques représentant un risque pour les consommateurs. Par ailleurs, la production possible d'huîtres tétraploïdes dans les installations d’élevage implique l’étude de solutions de traitement adaptées pour éviter tout rejet de gamètes, larves ou naissain d’animaux polyploïdes dans l'environnement. Le projet DESIMER a donc pour objectif d’étudier et de comparer différentes techniques de désinfection de l’eau de mer : la chloration, l’ozonation et le rayonnement UV afin de définir quelles approches techniques sont les plus adaptées tout en prenant en compte la création éventuelle de sous-produits de désinfection. Ainsi, trois techniques de désinfection de l’eau ont été ciblées : - la chloration, étudiée comme solution de référence car mise en œuvre dans de nombreuses structures aquacoles pour des opérations de nettoyage et de traitement des eaux intégrant différentes installations d’élevage de l’Ifremer; - l'ozonation, mise en œuvre notamment dans quelques grands aquariums français; - l'irradiation UV, largement utilisée à plusieurs étapes des circuits d'eau de mer alimentant les installations d’élevage d’écloseries privées et de la plateforme expérimentale de Bouin. Ce procédé présente l'avantage a priori de ne pas créer de sous-produits. Les travaux ont donc porté sur l’adaptation de ces différentes techniques à l’échelle du laboratoire et sur la mise au point des méthodes d’analyses (cytométrie en flux, biologie moléculaire –qPCR) et de suivi des performances des désinfections pour inactiver les pathogènes, les gamètes et larves de l’huître creuse Crassostrea gigas. Un suivi des sous-produits de désinfection a aussi été réalisé pour les différents traitements afin de les qualifier et de les quantifier. Les doses pour les trois techniques ont été déterminées afin de satisfaire ces objectifs en faisant appel à des outils et méthodologies fiables permettant de vérifier les performances de ces techniques. De plus, la question de la création de sous-produits de désinfection a été traitée et a permis de confirmer la formation de substances telles que le bromoforme après chloration et ozonation de l’eau de mer.

French production of the Pacific cupped oyster, Crassostrea gigas, is currently threatened by two pathogens, OsHV-1 and V. aestuarianus. While oysters selected for their higher resistance to OsHV-1 are now available for the industry, the impact of V. aestuarianus on such oysters is unknown, especially for triploids. In addition, experimental infection has used the virus or the bacteria alone, but there have been no investigations of dual exposure to these pathogens. This study is the first report of single or dual exposure in spat (Spat1 and Spat2), juvenile and adult naïve oysters. For each of the two stocks evaluated, unselected oysters and oysters selected for their higher resistance to OsHV-1 infection were tested, as well as their triploid siblings of the selected oysters produced using cytochalasin B. We confirmed that resistance to OsHV-1 infection and susceptibility to V. aestuarianus increased with age and size, although selected oysters were not significantly impacted by OsHV-1 whatever their ploidy, size or age. We found different mortality patterns depending on the pathogen tested. The mortality pattern was similar for oysters exposed to OsHV-1 or to both pathogens in the Spat1 trial (4 months old and 1.9 g). The mortality pattern was similar for oysters exposed to V. aestuarianus or to both pathogens in the Adult trial (25 months old and 63.1 g). Surprisingly, mortality was much higher (ranging from 75.9% to 100%), in particular for the selected oysters, for the Spat2 (8 months old/3.9 g) and Juvenile trials (16 months old/18.4 g) given a dual exposure, regardless of the level of selection for OsHV-1 and the ploidy state. Our findings highlight an important threat for oyster farmers: oysters exposed to both pathogens could experience dramatic mortality rates, even in oysters selected for their higher resistance to OsHV-1. Finally, our study demonstrated for the first time that triploid oysters were more susceptible to experimental challenges with V. aestuarianus at the spat stage than their diploid siblings. However, the difference in mortality between the triploids and diploids remained limited and ranged from 22.9% to 6.6% for spat and adults, respectively with a relatively regularly decrease in the difference with increased age.

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate. (c) 2008 Elsevier GmbH. All rights reserved.

Mass mortality outbreaks of Pacific oysters, Crassostrea gigas, are reported in different areas around the world affecting seriously the shellfish aquaculture sector. Two pathogens are associated with these mortality outbreaks, ostreid herpesvirus 1 (OsHV-1) and Vibrio aestuarianus. In this contexte, a better knowledge is needed in terms of oyster immunity. For this purpose, as an important degradation pathway autophagy was investigated in the Pacific oysters. An in silico research of genes involved in autophagy using the C. gigas genome allowed identification of homologs of ATG1 and ATG8/LC3(Microtubule-associated protein 1A/1B-light chain 3). Gene expression and protein detection were analysed using real-time PCR and western blot, respectively. ATG1 and ATG8 gene expression was upregulated during an experimental viral infection. Western blot analysis showed an increase of LC3 protein during the infection, suggesting an activation of autophagy. Ammonium chloride treatment was associated with increased oyster mortality whereas less mortality was reported after carbamazepine treatment in experimentally infected animals. Results suggest a protective role of autophagy against OsHV-1 and V. aestuarianus infection. This study contributes to better understand the innate immune system of Pacific oysters.

The ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus in the brackish aquatic environment was extensively investigated in this study. By conducting laboratory experiments under natural setting conditions, it was shown that V. splendidus LGP32 strain generally exhibits longer persistence in both seawater and sediment than V. aestuarianus 01/32 strain. Both strains maintained viability and culturability for longer times in the sediment, suggesting that this compartment may represent a suitable niche for their persistence in the environment. In addition, both strains attached to chitin particles and copepods, the efficiency of attachment being higher in V. splendidus than in V. aestuarianus. Similarly, LGP32 strain showed a greater capability to form biofilm on poly-vinyl chloride (PVC) surfaces than 01/32 strain. LGP32 and 01/32 strains were also capable of entering a viable but non-culturable state after extended incubation at 5°C, a condition commonly found during cold season in the aquatic brackish environment. These results are consistent with field data collected during a 2-year sampling campaign in the northern Adriatic Sea and provide background information on the mechanisms promoting V. splendidus and V. aestuarianus persistence in coastal water, thus contributing to a better understanding of the epidemiology of the associated diseases.

The complex interactions occurring between farmed bivalves and their potential pathogens in the circumstances of global climate changes are current matter of study, owing to the recurrent production breakdowns reported in Europe and other regions of the world. In the frame of Project FP7-KBBE-2010-4 BIVALIFE, we investigated the occurrence of mortality and potential pathogens during the Spring–Summer transition in Crassostrea gigas and Mytilus galloprovincialis cohabiting in the shallow waters of one northern Italian lagoon (Sacca di Goro, Adriatic Sea) and regarded as susceptible and resistant species, respectively. In 2011, limited bivalve mortality was detected in the open-field trial performed with 6–12 month old spat whereas subsequent trials with 2–3 month old spat produced almost complete (2012) and considerable (2013) oyster mortality. Macroscopical examination and histology excluded the presence of notifiable pathogens but, in the sampling preceding the massive oyster spat mortality of 2012, a μdeleted variant of OsHV-1 DNA was found in wide-ranging amounts in all analyzed oysters in conjunction with substantial levels of Vibrio splendidus and Vibrio aestuarianus. The large oyster spat mortality with borderline OsHV-1 positivity recorded in 2013 supports the multi-factorial etiology of the syndrome. This is the first report of a OsHV-1 (under a form interpreted as the variant μVar) in the Goro lagoon. Transcriptional host footprints are under investigation to better understand the bivalve response to environmental factors, included viral and bacterial pathogens, in relation to the observed mortalities.

Vibrio aestuarianus is frequently found in coastal areas and can infect and induce mortalities in the pacific oyster Crassostrea gigas. However, nothing is known about its distribution and seasonality in the estuarine environment, especially where oyster farming is practiced. Its occurrence was investigated in sediment and oyster haemolymph at 2 oyster farms in Brittany (France) over 2 yr during 2 periods, from June to September 2007 and from February to June 2008. Total heterotrophic bacteria (HB) were cultured on marine agar while total Vibrio spp. and V. aestuarianus were selectively numerated using thiosulfate citrate bile salts sucrose agar (TCBS agar) and the species-specific hybridisation method, respectively. PCR was performed to detect V. aestuarianus in sediment when it became unculturable. Both total Vibrio spp. and V. aestuarianus had a seasonal trend. The highest concentrations were recovered in the warmest months. Its abundance ranged from 10(2) to 4 x 10(5) CFU ml(-1) in haemolymph and from 10(3) to 1 x 10(4) CFU g(-1) in the sediment. Temperature was the main factor influencing the concentration of Vibrio spp. and V. aestuarianus in the sediment. Thus V aestuarianus might subsist during the cold seasons in the sediment, from which it can emerge when environmental conditions became favourable.

Since several years, the French oyster farming copes with high mortality affecting juveniles and some adults of the Pacific oyster Crassostrea gigas. Studies conducted in the framework of a multidisciplinary project (MOREST) showed that mortality was related to several factors like physiological and genetic status of oyster, pathogens including bacteria of the genus Vibrio, and revealed that the sediment, on which the oyster cages are installed, might play a particular role. This has been suggested because of the concomitance of the mortalities and increased toxic compounds like ammonia and sulphides in the sediment. Moreover, the presence of Vibrio bacteria in the sediment indicates a dual role of sediment in the mortality onset. The physico-chemical parameters, the structure of bacterial communities and the abundance of Vibrio aestuarianus, pathogen of C. gigas, have been determined in sediments below oyster tables and outside tables of the Aber Benoit (Nord-Finistère, French Atlantic coast) and Auray River (Gulf of Morbihan) in different seasons for 2 years. This work confirms the existence of a role of sediment risk and also the influence of oyster tables on sediment biogeochemistry. Indeed, high concentrations of ammonia and sulphides have been recorded in sediments under the tables of both sites. In the AberBenoît, the impact of oyster tables on the sediment biogeochemistry was more evident whereas the bacterial community structure showed certain stability. The microflora of sediments of the Auray River appeared more dynamic and was influenced by external inputs. These results demonstrate the importance of the characteristics related to each site in the response of bacterial communities following a disturbance. Furthermore, members of Vibrionaceae group were scarce in the sediments. Partial 16S rRNA gene sequences of two pathogenic species, namely V. ordalii and V aestuarianus, were detected, suggesting that they were part of the dominant community in the sediment. The study of the ecology of V. aestuarianus showed that it was present in the sediment during cold and warm seasons. However, presence of V. aestuarianus in sediment was not correlated to its abundance in oysters. Experiments conducted in microcosms with a GFP-transformed strain of V. aestuarianus showed that this bacterium could easily circulate between the sediment, the seawater and oysters and induce mortalities. Our results highlight the important of sediment as a reservoir of V. aestuarianus., Depuis plusieurs années, l’ostréiculture française doit faire face à de fortes mortalités qui touchent les juvéniles et parfois les adultes de l’huître creuse Crassostrea gigas. Les études réalisées dans le cadre d’un projet multidisciplinaire (MOREST) ont montré que les mortalités survenaient suite à l’interaction de plusieurs facteurs à savoir, l’état physiologique de l’huître et ses caractéristiques génétiques, des pathogènes parmi lesquels des bactéries du genre Vibrio, et l’environnement dont la température et le sédiment. Le rôle du sédiment a été suggéré du fait de la concomitance entre les mortalités, et l’augmentation des composés toxiques comme l’ammonium et les sulfures dans le sédiment. Par ailleurs, il pouvait constituer un réservoir de vibrions selon des études précédentes. Pour vérifier ces hypothèses les caractéristiques physico-chimiques, la structure des communautés bactériennes ainsi que l’abondance de V. aestuarianus, pathogène de C. gigas ont été déterminées dans les sédiments sous tables et hors tables des sites situés dans l’aber Benoît et en rivière d’Auray pendant 2 années. Ce travail a confirmé l’existence d’un risque sédimentaire par la présence de fortes concentrations d’ammonium et de sulfures dans les sédiments à certaines périodes de l’année ainsi que l’influence des huîtres sur leur intensité. Cependant, l’impact des tables ostréicoles sur ces flux n’est évident que dans l’aber Benoît. Il a mis en évidence une certaine stabilité de la structure et de la diversité des microflores des sédiments au regard des nombreuses influences qu’elles subissent, spécialement dans l’aber Benoît. La microflore des sédiments de la rivière d’Auray semble quant à elle plus dynamique et influencée par les apports extérieurs. Ces résultats prouvent l’importance des caractéristiques liées aux sites dans la réponse des communautés bactériennes face des modifications du milieu. La population de Vibrio est apparue peu diversifiée et peu abondante fluctuant avec la température comme les autres groupes bactériens. Deux espèces pathogènes semblent pourtant faire partie de la communauté dominante des sédiments à savoir V. ordalii et V aestuarianus. L’étude de l’écologie de ce dernier a montré qu’il est présent dans le sédiment pendant les saisons froide et chaude alors que sa détection dans les huîtres est plutôt occasionnelle. Des expérimentations en microcosmes avec la souche 02/041 transformée par un plasmide codant pour la GFP ont montré que cette bactérie pouvait circuler aisément entre le sédiment, l’eau de mer et les huîtres et provoquer des mortalités. Nos résultats mettent ainsi en évidence le rôle du sédiment en tant que réservoir de V. aestuarianus.

In summer 2006 an extensive mortality of Pacific oysters Crassostrea gigas occurred in Lake Grevelingen, the Netherlands. A sample of Pacific oysters was investigated for the presence of shellfish pathogens as potential causes of the mortality. Yellow-green lesions were observed in several oysters upon clinical inspection. Histopathology showed that 6 out of 36 oysters had a suspected bacterial infection, including 4 Nocardia-like infections. Two bacterial species, Vibrio aestuarianus and Nocardia crassostreae, were isolated from haemolymph samples and identified using PCR and sequencing of the 16S rRNA gene. This is the first isolation of N. crassostreae from shellfish in European waters. The near full-length 16S rRNA sequence of this Dutch Nocardia sp. isolate was identical to other known N. crassostreae isolates from the west coast of North America. The primary cause of oyster mortality was thought to be the physiological stress from environmental conditions, including prolonged high water temperatures and low oxygen levels. The multiple bacterial species isolated from the diseased Pacific oysters may have been a secondary cause.

Depuis une quinzaine d'années, des mortalités massives d'huîtres creuses Crassostrea gigas, sont observées sur le littoral français durant la période estivale. Des études ont démontré que les mortalités sont plurifactorielles et qu'elles sont la résultante d'interactions entre l'environnement, l'huître (physiologie et génétique) et certains agents infectieux. Durant ce stage, les interactions entre l'huître creuse et deux bactéries de genre Vibrio (Vibrio splendidus et Vibrio aestuarianus), fréquemment associées aux mortalités, ont été étudiées, afin de tenter de comprendre le lien entre les facteurs de l'hôte (origine génétique et physiologie de la reproduction) et les vibrions (production d'un facteur de virulence), pouvant impacter la sensibilité de l'huître creuse, diploïde et triploïde, soumise à une infection expérimentale par des Vibrio. Des infections expérimentales ont été effectuées par injection dans le muscle adducteur d'un mélange de Vibrio splendidus et Vibrio aestuarianus, sur des huîtres de ploïdie différente (diploïdes et triploïdes) et à différentes périodes de l'année correspondant chacune à un stade de reproduction distincts. Des animaux aux réponses contrastées à ces infections expérimentales (morts et survivants) ont été obtenus et caractérisés: mesure d'occupation gonadique par histologie, calcul de l'indice de condition par pesées et déterminisme de la contribution de géniteurs mâles sur la descendance diploïde et triploïde par génotypage. De plus, des expériences sur les deux pathogènes ont été menées afin d'étudier leur croissance et ainsi que leur capacité de production de métalloprotéase in vitro (seul facteur de virulence connu à ce jour chez ces deux vibrio). Dans un premier temps, nous avons pu voir que la croissance des deux pathogènes est différentes au cours du temps et que l'expression de métalloprotéases semble être moduler par un phénomène de quorum-sensing. Dans un second temps, nous avons pu démontrer que les huîtres diploïdes présentent une sensibilité plus importante durant la période de reproduction, alors que durant la période hivernale, les huîtres triploïdes sont plus sensibles que les diploïdes. Il est néanmoins probable que l'état de santé insuffisant des animaux triploïdes explique ce phénomène. Enfin une composante génétique à la résistance de l'huître a été mise en évidence, celle-ci paraissant s'exprimer différemment en fonction de la ploïdie. Pour conclure sur la problématique de ce stage, cette étude a permis de montrer qu'il n'existe probablement pas un seul facteur susceptible d'expliquer à lui seul la sensibilité de l'huître à une vibriose, même si chacun l'influence. Reproduction, état de santé, ploïdie et composante génétique semblent agir en interaction, ce qui conforte l'idée d'un phénomène plurifactoriel.

Les bactéries du genre Vibrio représentent le groupe le plus fréquemment associé à des maladies infectieuses chez les mollusques marins et notamment l'huître creuse Crassostrea gigas. A ce jour trois espèces de Vibrio pathogènes d'huîtres creuses V. splendidus, V. aestuarianus et récemment V. coralliitycus ont été identifiés en France. La prévention des mortalités associées à ces vibrioses repose sur une meilleure compréhension des interactions hôte-pathogène et sur l'utilisation de techniques de diagnostic rapides à mettre en oeuvre et fiables. Ce travail a permis d'une part de caractériser la dynamique de phagocytose de vibrions par les cellules immunocompétentes de l'huître, les hémocytes. La mise au point d'un protocole d'analyse en cytométrie de flux des constituants de l'hémolymphe révèle l'existence de grandes variations de la capacité de phagocytose des huîtres dont la cause est discutée. D'autre part des tests de diagnostic moléculaire qualitatifs (PCR classiques) et quantitatifs (PCR quantitative) permettant de détecter spécifiquement les trois vibrions pathogènes rencontrés chez l'huître creuse ont été développés et sont en cours d'optimisation.

In France. shellfish production is a traditional. well-established industry. mainly based upon commercial farming of the Pacifie oyster. Crassostrea gigas. Since the 1980's, annual mass mortalities of C. gigas have been reported during summer. Several studies on this subject have demonstrated that these mortality outbreaks resulted from complex interactions between the physiologieal andJor genetic status of the oysters. environmental factors, and one or more infectious agents, among which are bacteria of the genus Vibrio., En France, l'ostréiculture constitue la première production de mollusques et repose essentiellement sur l'huître creuse du Pacifique ou huître japonaise Crassostrea gigas. Depuis les années 80, les ostréiculteurs doivent toutefois faire face à des épisodes de mortalité de certains cheptels lors de la période estivale. Les études réalisées dans le cadre d'un programme de recherche multidisciplinaire ont établi que ce phénomène résultait d'une interaction complexe entre le statut physiologique et/ou génétique 'de l'huître, des paramètres environnementaux particuliers, et la présence de microorganismes potentiellement pathogènes, dont des bactéries du genre Vibrio.