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The parasite Haplosporidium costale is known to infect and cause mortality in the oyster Crassostrea virginica in the USA. Decades after its first description in the 1960s, this parasite was detected in Crassostrea gigas in the USA and China. However, it presented a low prevalence and no mortality was associated with it. More recently, in 2019, H. costale was detected in France in a batch of moribund oysters. In order to observe how long this parasite has been present on French coasts, from Normandy to Thau lagoon, a retrospective investigation was conducted on 871 adult and spat oyster batches from 2004 to 2020. To allow rapid detection on a large panel of samples, a real-time PCR for the H. costale actin gene was developed. This method allowed the detection of H. costale DNA in adults from 2005 and in spat from 2008. The H. costale prevalence in spat appeared higher than in adults over the years studied, 14.59 % compared to 6.50 %, respectively. All samples presenting positive results were then sequenced on two targets, H. costale rRNA and actin genes. The actin gene sequencing highlighted the presence of two H. costale strains. Adult C. gigas as well as spat batches coming from hatcheries and DNA controls from C. virginica all presented with the Profile 1 H. costale strain. The Profile 2 H. costale strain was detected only in C. gigas spat coming from natural sources. These observations suggest a correlation between the origin of oysters and H. costale strains which may have been caused by commercial imports between Japan, USA and France back to the 1970s. Over the positive samples studied, only few batches (n = 3) suffered mortalities which could be hypothesized to be caused by H. costale, all presenting the Profile 1 H. costale strain.

International audience; Since 2010, mass mortality events known as Pacific oyster mortality syndrome (POMS) have occurred in Crassostrea gigas in Australia associated with Ostreid herpesvirus 1. The virus was thought to be an OsHV-1 µVar or "microvariant", i.e. one of the dominant variants associated with POMS in Europe, but there are few data to characterize the genotype in Australia. Consequently, the genetic identity and diversity of the virus was determined to understand the epidemiology of the disease in Australia. Samples were analysed from diseased C. gigas over five summer seasons between 2011 and 2016 in POMS-affected estuaries: Georges River in New South Wales (NSW), Hawkesbury River (NSW) and Pitt Water in Tasmania. Sequencing was attempted for six genomic regions. Numerous variants were identified among these regions (n = 100 isolates) while twelve variants were identified from concatenated nucleotide sequences (n = 61 isolates). Nucleotide diversity of the seven genotypes of C region among Australian isolates (Pi 0.99 × 10 − 3) was the lowest globally. All Australian isolates grouped in a cluster distinct from other OsHV-1 isolates worldwide. This is the first report that Australian outbreaks of POMS were associated with OsHV-1 distinct from OsHV-1 reference genotype, µVar and other microvariants from other countries. The findings illustrate that microvariants are not the only variants of OsHV-1 associated with mass mortality events in C. gigas. In addition, there was mutually exclusive spatial clustering of viral genomic and amino acid sequence variants between estuaries, and a possible association between genotype/amino acid sequence and the prevalence and severity of POMS, as this differed between these estuaries. The sequencing findings supported prior epidemiological evidence for environmental reservoirs of OsHV-1 for POMS outbreaks in Australia.

The current study describes the development and application of a TaqMan® real-time PCR assay for the detection of the bacterium Francisella halioticida. Previously, detection of F. halioticida is relied on bacterial culture and conventional PCR; however, the real-time PCR provides many advantages because it is faster, less labour-intensive and reduces the risk of cross-contamination. DNA samples from mussels collected in April 2020 from seven sites in northern Brittany (France) were tested using the newly developed real-time PCR assay. The objective was to screen for the presence of F. halioticida during spring mortality events. The bacterium was detected in 71.4% of the samples tested and was present at all sites except for Saint-Brieuc and Mont-Saint-Michel, two sites which were not concerned by mortality at the time of sampling. Less than a month later, Saint-Brieuc was affected by unusual mortalities and F. halioticida was detected in almost all mussels (81.25%). The findings from this study provide further evidence indicating that F. halioticida may be contributing to mussel mortalities; however, a direct causal relationship has not yet been established. The real-time PCR assay developed in this study allows for rapid, specific and sensitive detection of F. halioticida which should prove useful for future studies concerning the involvement of this bacterium with shellfish mortalities.

Transmissible cancers are parasitic malignant cell lineages that have acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukaemia-like disease. In Mytilus mussels, two lineages of bivalve transmissible neoplasia (BTN) have been described to date (MtrBTN1 and MtrBTN2), both of which emerged in a Mytilus trossulus founder individual. Here, we performed extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping 106single nucleotide polymorphisms of 5,907 European Mytilus mussels. Genetic analysis allowed us to simultaneously obtain the genotype of hosts - Mytilus edulis, M.galloprovincialis or hybrids - and the genotype of tumours of heavily infected individuals. In addition, a subset of 222 individuals were systematically genotyped and analysed by histology to screen for possible nontransmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M.galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found, but eight individuals with nontransmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M.trossulus individuals. The observed polymorphism is probably due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two sublineages divergent by 10% fixed somatic null alleles and one nonsynonymous mtCOI (mitochondrial cytochrome oxidase I) substitution are cospreading in the same geographical area, suggesting a complex diversification of MtrBTN2since its emergence and host species shift.

Transmissible cancers are parasitic malignant cell lineages that acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukemia-like disease. In Mytilus mussels, two lineages of Bivalve Transmissible Neoplasia (BTN), both emerged in a M. trossulus founder individual, have been described to date (MtrBTN1 and MtrBTN2). Here, we performed an extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping hundred SNPs of thousands of European Mytilus mussels. The genetic analysis allowed us to simultaneously obtain the genotype of hosts -M. edulis, M. galloprovincialis or hybrids- and the genotype of tumors of heavily infected individuals. In addition, a subset of individuals were systematically genotyped and analysed by histology in order to screen for possible non-transmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M. galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found but a few individuals with non-transmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2 genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M. trossulus individuals. The observed polymorphism is most likely due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two divergent sublineages, confirmed by mtCOI sequences, are co-spreading in the same geographic area, suggesting a complex diversification of MtrBTN2 since its emergence and host species shift.

International audience; Mortality outbreaks in adult oysters (Crassostrea gigas) have been reported since 1994 in Normandy. Recent work has shown that triploid oysters tend to be subject to higher mortality than diploids. Many parameters could be involved in this phenomenon including pathogens, environmental conditions, the physiological and genetic states of the oysters. With respect to reproductive physiology, studies showed that triploid oysters have gamete production. In order to understand the abnormal mortalities, we focused on gametogenesis and more specifically on hemocyte infiltration in gonadal tubules. Fourteen batches of oysters with different origins and ploidy were followed during two years. The results of the study showed intense hemocyte infiltration in gonadal tubules during the ripe stage of gametogenesis in triploid oysters. Finally, this article suggests a link between the frequency of individuals with hemocyte infiltration in gonadal tubules and the proportion of mortality within the triploid groups.

Transmissible cancers, in which cancer cells themselves act as an infectious agent, have been identified in Tasmanian devils, dogs, and four bivalves. We investigated a disseminated neoplasia affecting geographically distant populations of two species of mussels (Mytilus chilensis in South America and M. edulis in Europe). Sequencing alleles from four loci (two nuclear and two mitochondrial) provided evidence of transmissible cancer in both species. Phylogenetic analysis of cancer-associated alleles and analysis of diagnostic SNPs showed that cancers in both species likely arose in a third species of mussel (M. trossulus), but these cancer cells are independent from the previously identified transmissible cancer in M. trossulus from Canada. Unexpectedly, cancers from M. chilensis and M. edulis are nearly identical, showing that the same cancer lineage affects both. Thus, a single transmissible cancer lineage has crossed into two new host species and has been transferred across the Atlantic and Pacific Oceans and between the Northern and Southern hemispheres., eLife digest Cancer cells can grow and spread in one individual, but they normally do not spread to others. There are a few exceptions to this rule. For example, there are cancers in Tasmanian devils, dogs and bivalve shellfish that can spread to other members of the same species. In these creatures, cancer from one individual evolved the ability to spread throughout the population. These cancer cells infect animals like a pathogen. A fatal cancer called disseminated neoplasia affects many species of bivalves. In four bivalve species, including the marine mussel Mytilus trossulus, scientists have shown that the cancer can spread from one individual to another. This transmissible cancer has been found in M. trossulus mussels in British Columbia, Canada; but related species of mussels in other parts of the world also develop disseminated neoplasia. It is possible these other cancers are transmissible and have spread from one population of mussels to another. Yonemitsu et al. performed genetic analyses to show that cancers found in two other mussel species – Mytilus chilensis in South America and Mytilus edulis in Europe – are transmissible and arose in M. trossulus. The cancers in the South American and European mussels were nearly identical genetically, which suggests that they came from a single M. trossulus mussel with cancer at some point in the past. Somehow cancer cells spread between the Northern and the Southern Hemispheres and across the Atlantic Ocean, infecting multiple species across the world. The analyses also show that this cancer lineage is different from the one previously identified in British Columbia. These analyses show that bivalve transmissible neoplasia was able to spread worldwide, most likely through accidental transport of infected mussels on international shipping vessels. This suggests that human activities unwittingly introduced the disease to new areas. Learning more about transmissible cancers may help scientists understand how cancers evolve with their hosts in extreme situations.

International audience; This study aimed at assessing the pathogenicity of two Vibrio splendidus‐related species and evaluating the influence of the origin and annual life cycle of mussels on their sensitivity during a bacterial challenge. Thus, in vivo infection assays were made with Vibrio crassostreae 7T4_12 and Vibrio splendidus 3G1_6, over, respectively, thirteen and 9 months, on adult blue mussels from five recruitment areas in France. Two bacterial concentrations were tested: one consistent with the loads of Vibrio spp. in environment and mussel tissues (~105 CFU/ml) and another one much higher (~108 CFU/ml). The tested environmental concentration has no pathogenic effect whatever the time of year, the strain used and the origin of mussels. However, at the highest concentration, a pathogenic effect was observed only at specific moments, and one of the origins appeared to be more resistant. The physiological state of mussels—depending on the time of year—seemed significant in mussels’ sensitivity, as their recruitment origin. This study is the first to test the pathogenicity of V. splendidus‐related strains at concentrations close to what is found in the wild, over the annual cycle of mussels, and considering their origin.

International audience; Marine mussel production is of substantial economic interest in numerous coastal areas worldwide, making crucial the study of pathologies that affect them. Disseminated neoplasia (DN) has recently been suggested to be linked to blue mussel, Mytilus edulis, mortality outbreaks observed in France since 2014, although the evidence remains indirect. In order to improve DN detection and monitoring, we compared the sensitivity of four diagnostic tools, namely haemocytology, histology, flow cytometry, and genetics. Haemocytological examination gave the best results in sensitivity and had the advantage of being non-invasive, allowing disease progression to be followed in affected mussels. Using this approach, we showed that DN progression is usually slow, and we provide evidence of remission events. We observed a high diversity of forms and mitotic features of neoplastic cells located in the vesicular connective tissue but rarely in the haemolymph. Circulating cells occur as four main types but are homogenous in morphology and DNA content within a single individual. Polyploidy proved very high, from 8 N to 18 N. Genetic analysis of haemolymph DNA showed that a Mytilus trossulus genetic signal was associated with almost all the DN cases here diagnosed by haemocytological examination, regardless of the DN type. This result corroborates DN is a transmissible cancer that first originated in a M. trossulus host and subsequently crossed into M. edulis. No pre-neoplastic conditions were detectable. The prevalence of the disease was quite low, which, together with the low morbidity observed in the lab, suggest DN is unlikely to be the direct cause of mortality outbreaks in France.

International audience; The Vibrio splendidus species is ubiquitous in the marine environment but have also been recognized as pathogenic for several aquatic animals. This species belongs to the Splendidus clade which is composed of 16 genetically related species. To our knowledge, there are no available tools able to discriminate the V. splendidus species from the other members of the clade. Thus, we developed a Real Time PCR based on the toxR gene specific to V. splendidus species. Specificity tests were performed on 74 reference strains and 116 field strains and gave 96.05% inclusivity and 100% exclusivity results. The limit of detection of the PCR was determined according to the XP-U47-600-2 AFNOR norm and was estimated at 18 copies genome per reaction. This work describes a reliable, sensible and specific tool to discriminate V. splendidus species from other members of the Splendidus clade and thus could be clearly suitable to perform taxonomic investigations.

The acute course of disease in young oysters infected by OsHV-1 and the rapid tissue degradation often preclude histological examination of specimens collected during outbreaks in field. Herein, live spat originated from two geographical areas were sampled just at the onset of a mortality event that occurred in Normandy (France) in June 2016. The lesions, associated with high OsHV-1 DNA quantities, were characterized by severe and diffuse haemocytosis mainly involving blast-like cells, myocyte degeneration and large, irregularly shaped degenerate eosinophilic cells in the connective tissue. The herpesvirus was identified by negative staining TEM and real-time PCR. Sequencing of the C region and ORFs 42/43 confirmed that the variants met the definition of OsHV-1 μVar. We sequenced 30 other ORFs in twenty OsHV-1-positive individuals and compared them to the μVar specimens isolated between 2009 and 2011. The ORFs encoding putative membrane proteins showed the highest number of variations. Seven different genotypes were identified, confirming the presence of relevant genetic diversity. Phylogenetic analysis provided evidence for a well-separated μVar new group, with an evolutionary divergence estimated at 0.0013 from the other μVar variants. The geographical distribution of these newly described variants and their effective virulence should be investigated in future.

International audience; In 2014, a high and unusual mass mortality of mussels occurred in several important production areas along the French coasts of the Atlantic and English Channel. In the first quarter of 2016, mass mortalities hit farms on the west coast of the country once again. These heterogeneous mortality events elicited a multi-parametric study conducted during the 2017 mussel season in three sites in northern Brittany (Brest, Lannion and St. Brieuc). The objective was to assess the health status of these mussels, follow mortality and attempt to identify potential causes of the abnormal high mortality of farmed mussels in northern Brittany. Brest was the most affected site with 70% cumulative mortality, then Lannion with 40% and finally St. Brieuc with a normal value of 15%. We highlighted a temporal ‘mortality window’ that opened throughout the spring season, and concerned the sites affected by mortality of harmful parasites (including pathogenic bacteria), neoplasia, metal contamination, and tissue alterations. Likely, the combination of all these factors leads to a weakening of mussels that can cause death.

International audience; Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis are emerging pathogen parasites in the food domain. However, without standardized methods for their detection in food matrices, parasitic foodborne outbreaks remain neglected. In this study, a new immunomagnetic separation assay (IMS Toxo) targeting the oocyst's wall of T. gondii was developed using a specific purified monoclonal antibody. Performance of this IMS Toxo coupled to microscopic and qPCR analyses was evaluated in terms of limit of detection (LOD) and recovery rate (RR) on: i) simple matrix (LOD ¼ 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD ¼ 33 oocysts/g; RR between 0.2 and 35%). Finally, to simultaneously extract the three protozoa from these food matrices, T. gondii oocysts were directly concentrated (without IMS Toxo) from the supernatant of the IMS of Cryptosporidium and Giardia (oo)cysts. This strategy associated to qPCR detection led to LOD

International audience; Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection.Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level

International audience; This study evaluates the viability of Giardia intestinalis cysts, Cryptosporidium spp. and Toxoplasma gondii oocysts, which may be found in water and food matrices subjected to contaminated water. Viability is a key factor to be considered in order to assess parasite infectivity. People living in developing countries are particularly at risk of contamination by these pathogens, which can cause severe diseases particularly in immunocompromised people. In this report, we describe methods combining the mechanical rupture of (oo)cysts and mRNA extraction in order to determine their viability by qRT-PCR. The targeted genes are beta-giardin, hsp70, and SporoSAG for the detection of G. intestinalis, Cryptosporidium spp. and T. gondii respectively. The proposed method is compared to in vivo infectivity tests specific of each parasite. Then, this qRT-PCR method is applied directly on parasite suspensions and subsequently on basil experimentally contaminated with the parasites. For each protozoan, the detection limit of the qRT-PCR method is 2 parasites per reaction (2 μL mRNA analyzed) on parasite suspensions, and 3 parasites per gram of basil matrix. Compared to in vivo methods, qRT-PCR can detect viable but not infectious parasites. This qRT-PCR method could represent an attractive alternative as a specific and sensitive tool for: i) rapid assessment of the risk of human contamination by G. intestinalis, Cryptosporidium spp. and T. gondii parasites; ii) evaluation of the efficiency of industrial process.

Deux methodes appliquees a la recherche des virus enteriques dans les boues de station d'epuration ont ete etudiees. Dans un premier temps, les deux methodes, comprenant chacune une etape de concentration et une etape de detection par culture cellulaire, ont ete comparees entre elles. Dans un second temps, seules les techniques de concentration ont ete comparees en analysant les concentrats par RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). Trois types de virus enteriques (enterovirus, rotavirus, virus de l'hepatite A) ont ete recherches dans differentes boues (stabilisees ou non et chaulees ou non). Les resultats obtenus avec les deux methodes sont coherents. Toutefois, l'une d'entre elles, bien que legerement moins sensible, offre un certain nombre d'avantages, notamment en termes de facilite de mise en oeuvre et de delai de reponse. L'utilisation de la RT-PCR pour cette comparaison a permis de confirmer l'interet de cette technique en termes de cout et de rapidite. Elle constitue egalement une alternative interessante pour les virus qui se multiplient mal en culture cellulaire. Enfin, les resultats des differentes analyses realisees au cours de cette etude, et notamment les resultats de denombrement, montrent que le procede d'hygienisation des boues permet de diminuer l'infectiosite des enterovirus.

International audience; Geotrichum candidum is a micro-fungus widely used as a ripening starter in cheese making. In anthropogenic environments such as dairy industries, this microorganism is subjected to many environmental and technological stresses including low temperature exposure. Our aim was to study the proteomic response of G. candidum to cold stress using a comparative proteomic approach by two-dimensional Differential In Gel Electrophoresis (2D DIGE). This technique consists on the labeling of proteins by specific fluorescent dyes (CyDyes). The results, obtained with G. candidum cells subjected to cold temperature, show significant proteomic patterns differences compared with the standard conditions. Furthermore, this biochemical response seems strain specific. 2D DIGE technology combined with SameSpots™ software analysis support these results through an important statistical validity. The comparative studies in a single gel, using two different fluorescent CyDyes (Cy3 and Cy5), lead to proteins differentiation. Selected spots were treated and analyzed by mass spectrometry.

Genotoxic impact of the occupational exposure was measured in farmers from Normandy, France. White blood cell DNA-adduct levels were measured for 116 non-smoking French crop farmers, using the (32)P-postlabelling method. A single blood sample was collected per farmer, at a randomised period of the year. Significantly higher bulky DNA-adduct levels were observed for samples collected from April to July, compared with samples collected during the other months. Agricultural practices were not significantly different between these two groups of farmers, but interestingly, the mean and the median duration without exposure to pesticides were significantly shorter for farmers sampled between April and July. These data, obtained in a homogeneous population of farmers, indicate a genotoxic impact for a sub-group, with a potential association with the use of pesticides. From the rest of the group, this study also gives for the first time additional information on the background fluctuations of this biomarker over the year.

Since summer 2008, high mortality rates of young Pacific oysters Crassostrea gigas have been recorded in association with the detection of the Ostreid Herpesvirus 1 (OsHV-1). A new variant called μVar has been recently described, characterized mainly by 12 consecutive deletions followed by one deletion of an adenine in the C region. The purpose of this study is to characterize the genotype (variants or OsHV-1 reference) of 300 positive samples of C. gigas analyzed between July 2008 and July 2010 collected along the French, Jersey, and Irish coasts. Samples were quantified by TaqMan PCR, amplified with conventional PCR, targeting the area of the deletion, and then sequenced. Eighty-seven percent of the samples were characterized and the OsHV-1 μVar was detected in 257 oyster samples. The genotype OsHV-1 reference was never detected during the 25 months of the present survey. Thirty-eight samples could not be determined and the majority of them had a low viral load. A novel genotype containing only 9 consecutive deletions named OsHV-1 μVar Δ9 was found in 5 samples. These observations indicate the emergence of different OsHV-1 variants.

Background Massive mortalities have been observed in France since 2008 on spat and juvenile Pacific oysters, Crassostrea gigas. A herpes virus called OsHV-1, easily detectable by PCR, has been implicated in the mortalities as demonstrated by the results of numerous field studies linking mortality with OsHV-1 prevalence. Moreover, experimental infections using viral particles have documented the pathogenicity of OsHV-1 but the physiological responses of host to pathogen are not well known. Results The aim of this study was to understand mechanisms brought into play against the virus during infection in the field. A microarray assay has been developed for a major part of the oyster genome and used for studying the host transcriptome across mortality on field. Spat with and without detectable OsHV-1 infection presenting or not mortality respectively were compared by microarray during mortality episodes. In this study, a number of genes are regulated in the response to pathogen infection on field and seems to argue to an implication of the virus in the observed mortality. The result allowed establishment of a hypothetic scheme of the host cell’s infection by, and response to, the pathogen. Conclusions This response shows a “sensu stricto” innate immunity through genic regulation of the virus OsHV-1 life cycle, but also others biological processes resulting to complex interactions between host and pathogens in general.

L'auteur a souhaité limiter l'accès de ce document aux membres de l'Enseignement supérieur français; This internship focuses on the study of the behavior of the parasite Haplosporidium costale in the oyster Crassostrea gigas. Indeed, this parasite causes 25 to 50% of mortality in Crassostrea virginica in the United States. In France, it was found for the first time in C. gigas in march 2019 by IFREMER following the observation of mortality on these oysters. In order to study its behavior, as a first step, the development of a real-time PCR and an In Situ Hybridization (ISH) method was carried out. Then using these tools, the study of its prevalence and parasitic cycle in C. gigas was sought. With these methods, the presence of parasitic DNA in adult oysters dating from 2005 could be highlighted as well as its presence in spat. A major difference in the presence of parasitic DNA was found between spat from hatchery (35% DNA from H. costal) and natural uptake (0%). ISH allowed the observation of a structure resembling H. costale in a sample from december 2019. To confirm the discovered information, a study over one year with a larger sample would have to be done.; Ce stage porte sur l’étude du comportement du parasite Haplosporidium costale chez l’huître creuse Crassostrea gigas. En effet, ce parasite cause 25 à 50% de mortalité chez Crassostrea virginica aux Etats-Unis. En France, il a été retrouvé pour la première fois chez C. gigas en mars 2019 par l’IFREMER suite à l’observation de mortalité sur ces huîtres. Afin d’étudier son comportement, dans un premier temps, le développement d’une PCR en temps réel et d’une méthode d’Hybridation In Situ (HIS) a été effectuée. Puis à l’aide de ces outils, l’étude de sa prévalence et du cycle parasitaire chez C. gigas a été recherché. Avec ces méthodes, la présence de l’ADN parasitaire dans des huîtres adultes datant de 2005 a pu être mis en avant ainsi que sa présence dans des naissains. Une différence majeure dans la présence de l’ADN parasitaire a été trouvée entre des naissains provenant d’écloserie (35% d’ADN d’H. costale) et de captage naturel (0%). L’HIS a permis l’observation d’une structure ressemblant au plasmode d’H. costale dans un échantillon de décembre 2019. Pour permettre de confirmer les informations découvertes une étude sur une année avec un échantillonnage plus important serait à faire.

Since 2014, mussels from several production areas located on the Atlantic coast and along the English Channel have been affected by massive mortality events. As a result, many mussel farmers have experienced a drastic reduction in production following these unusual and unexplained mortalities. These events have disrupted a whole "coastal socio-ecosystem" and have generated questions and concerns from professionals in the mussel farming sector. Indeed, with more than 50,000 tons of mussels produced per year, France ranks third in Europe in terms of volume (FAO, 2018) and occupies the first place in terms of product value with an estimated annual turnover of ~130 million euros (Agreste, 2019); thus, mussel farming represents a significant part of the French economy. The objective of this PhD thesis is to contribute to the understanding of the phenomenon of mussel mortality by studying the pathogens, pathological conditions and physiological conditions of animals that may be more or less involved in mortality. This was done to provide additional knowle dge about different hypotheses that had been formulated during the first episodes of high mortality. Therefore, different approaches were used. First, an analytical epidemiological approach was used; initially as part of a follow-up study over several months of mussel mortalities in North Brittany, then concomitantly, in the case of mortality declarations in Normandy/Hauts-de-France. In fact, since mussel mortalities are unusual, heterogeneous and unexplained, without a specifically identified etiological agent, it seemed important to consider several biotic, abiotic, internal and external parameters of the host. Then, in a different strategy, attention was focused on studying the implication of different criteria, specific to individuals, on their sensitivity during experimental bacterial infections. And finally, in a process of monitoring a disease, hemocytic neoplasia, Normandy mussels had been followed for several months to assess the prevalence, evolution and incidence of the disease on observed mortality.; Depuis 2014, les moules de certains bassins de production situés sur la façade atlantique et le long des côtes de la Manche subissent des épisodes de surmortalités. En conséquence, nombre de mytiliculteurs ont ainsi vu leur production se réduire drastiquement à la suite de ces mortalités singulières et inexpliquées. Ces événements ont perturbé tout un « socio-écosystème littoral » et ont généré des questionnements et des inquiétudes de la part des professionnels du secteur. En effet, avec plus de 50 000 tonnes de moules produites par an, la France se place au troisième rang européen en termes de volume (FAO, 2018) et occupe la première place au regard de la valeur du produit avec un chiffre d’affaires annuel estimé à ~130 millions d’euros (Agreste, 2019) ; ainsi, la mytiliculture représente une part non négligeable de l’économie française. L’objectif de ce travail de thèse est de contribuer à la compréhension du phénomène de mortalité chez les moules en étudiant les pathogènes, les états pathologiques et les conditions physiologiques des animaux qui peuvent plus ou moins être impliqués dans les mortalités. Ceci a été réalisé en vue d’apporter des éléments de connaissance supplémentaires au sujet de différentes hypothèses qui avaient été formulées lors des premiers épisodes de surmortalité. Pour ce faire, différentes approches ont été employées. Dans un premier temps, une démarche épidémiologique analytique a été utilisée ; d’abord dans le cadre d’une étude de suivi sur plusieurs mois des mortalités de moules de Bretagne Nord, puis de manière concomitante, dans les cas de déclarations de mortalités en Normandie/Hauts-de-France. De fait, les mortalités de moules ayant un caractère inédit, hétérogène et inexpliqué, sans agent étiologique spécifiquement identifié, il apparaissait important de considérer plusieurs paramètres biotiques, abiotiques, internes et externes à l’hôte. Ensuite, dans une stratégie différente, l’attention s’est portée sur l’étude de l’implication de différents critères, propres aux individus, sur leur sensibilité lors d’infections expérimentales bactériennes. Et pour finir, dans une démarche de suivi d’une maladie, la néoplasie hémocytaire, des moules de Normandie ont été suivies pendant plusieurs mois afin d’évaluer la prévalence, l’évolution et l’incidence de la maladie sur les mortalités observées.

Since 2014, mussels from several production areas located on the Atlantic coast and along the English Channel have been affected by massive mortality events. As a result, many mussel farmers have experienced a drastic reduction in production following these unusual and unexplained mortalities. These events have disrupted a whole "coastal socio-ecosystem" and have generated questions and concerns from professionals in the mussel farming sector. Indeed, with more than 50,000 tons of mussels produced per year, France ranks third in Europe in terms of volume (FAO, 2018) and occupies the first place in terms of product value with an estimated annual turnover of ~130 million euros (Agreste, 2019); thus, mussel farming represents a significant part of the French economy. The objective of this PhD thesis is to contribute to the understanding of the phenomenon of mussel mortality by studying the pathogens, pathological conditions and physiological conditions of animals that may be more or less involved in mortality. This was done to provide additional knowle dge about different hypotheses that had been formulated during the first episodes of high mortality. Therefore, different approaches were used. First, an analytical epidemiological approach was used; initially as part of a follow-up study over several months of mussel mortalities in North Brittany, then concomitantly, in the case of mortality declarations in Normandy/Hauts-de-France. In fact, since mussel mortalities are unusual, heterogeneous and unexplained, without a specifically identified etiological agent, it seemed important to consider several biotic, abiotic, internal and external parameters of the host. Then, in a different strategy, attention was focused on studying the implication of different criteria, specific to individuals, on their sensitivity during experimental bacterial infections. And finally, in a process of monitoring a disease, hemocytic neoplasia, Normandy mussels had been followed for several months to assess the prevalence, evolution and incidence of the disease on observed mortality.; Depuis 2014, les moules de certains bassins de production situés sur la façade atlantique et le long des côtes de la Manche subissent des épisodes de surmortalités. En conséquence, nombre de mytiliculteurs ont ainsi vu leur production se réduire drastiquement à la suite de ces mortalités singulières et inexpliquées. Ces événements ont perturbé tout un « socio-écosystème littoral » et ont généré des questionnements et des inquiétudes de la part des professionnels du secteur. En effet, avec plus de 50 000 tonnes de moules produites par an, la France se place au troisième rang européen en termes de volume (FAO, 2018) et occupe la première place au regard de la valeur du produit avec un chiffre d’affaires annuel estimé à ~130 millions d’euros (Agreste, 2019) ; ainsi, la mytiliculture représente une part non négligeable de l’économie française. L’objectif de ce travail de thèse est de contribuer à la compréhension du phénomène de mortalité chez les moules en étudiant les pathogènes, les états pathologiques et les conditions physiologiques des animaux qui peuvent plus ou moins être impliqués dans les mortalités. Ceci a été réalisé en vue d’apporter des éléments de connaissance supplémentaires au sujet de différentes hypothèses qui avaient été formulées lors des premiers épisodes de surmortalité. Pour ce faire, différentes approches ont été employées. Dans un premier temps, une démarche épidémiologique analytique a été utilisée ; d’abord dans le cadre d’une étude de suivi sur plusieurs mois des mortalités de moules de Bretagne Nord, puis de manière concomitante, dans les cas de déclarations de mortalités en Normandie/Hauts-de-France. De fait, les mortalités de moules ayant un caractère inédit, hétérogène et inexpliqué, sans agent étiologique spécifiquement identifié, il apparaissait important de considérer plusieurs paramètres biotiques, abiotiques, internes et externes à l’hôte. Ensuite, dans une stratégie différente, l’attention s’est portée sur l’étude de l’implication de différents critères, propres aux individus, sur leur sensibilité lors d’infections expérimentales bactériennes. Et pour finir, dans une démarche de suivi d’une maladie, la néoplasie hémocytaire, des moules de Normandie ont été suivies pendant plusieurs mois afin d’évaluer la prévalence, l’évolution et l’incidence de la maladie sur les mortalités observées.