Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

First isolation of Francisella halioticida strains from blue mussel (Mytilus edulis) in Normandy, France.

Mass mortality events affecting the blue mussels Mytilus edulis have been observed in France since 2014. The DNA of the bacterium Francisella halioticida, reported as pathogen of giant abalone (Haliotis gigantea) and Yesso scallop (Mizuhopecten yessoensis) has been detected recently in mussels from areas suffering mortalities. Isolation of this bacterium was attempted from individuals collected during mortality events. Identification was performed by 16S rRNA gene sequencing, real-time specific PCR and MALDI-ToF using spectra produced from the strain 8472-13A isolated from diseased Yesso scallop in Canada. Five isolates were identified as F. halioticida by real-time specific PCR and 16S rRNA sequencing. MALDI-ToF allowed the direct identification of four isolates (FR22a,b,c,d) which had 100% identity on the 16S rRNA gene with the known strains. On the other hand, one isolate (FR21) was not recognized by MALDI-ToF and had 99.9% identity on the 16S rRNA gene. The FR22 isolates showed difficult growth and required media optimization, which was not the case with the FR21 isolate. For these reasons, it was hypothesized that two type strains are present on French coasts, named FR21 and FR22. The FR21 isolate was selected for phenotypic analysis (growth curve, biochemical characteristics, electron microscopy), phylogenetic analysis and an experimental challenge. This isolate showed distinct differences compared to published F. halioticida strains, both at phenotypic and genotypic levels. Experimental infections of adult mussels led to 36% mortalities in 23 days following intramuscular injection with 3 × 107 CFU while a lower dose (3 × 103 CFU) did not lead to significant mortalities. In the context of this study, the strain FR21 was not virulent towards adult mussels.

Diversity and molecular epidemiology of Ostreid herpesvirus 1 in farmed Crassostrea gigas in Australia: Geographic clusters and implications for "microvariants" in global mortality events.

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.

Detection of the protistan parasite, Haplosporidium costale in Crassostrea gigas oysters from the French coast: A retrospective study.

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.

Prevalence and polymorphism of a mussel transmissible cancer in Europe.

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 106 single 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 MtrBTN2 genotypes 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 MtrBTN2 since its emergence and host species shift.

Development of a semi-quantitative PCR assay for the detection of Francisella halioticida and its application to field samples.

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.

First detection of Francisella halioticida in mussels Mytilus spp. experiencing mortalities in France.

This note describes the first detection of the bacteria Francisella halioticida in mussels Mytilus spp. from locations in Normandy and northern Brittany (France) experiencing high mussel mortalities, while it was not detected in the Bay of St Brieuc (northern Brittany), an area which was not affected by abnormal mussel mortality. The distribution of the bacteria in mussels seems to be restricted to inflammatory granulomas as observed in Yesso scallops Mizuhopecten yessoensis from Canada and Japan. F. halioticida has been identified as being involved in mass (>80%) mortality of abalones Haliotis gigantea in Japan and high (up to 40%) mortality of Yesso scallops Mizuhopecten yessoensis in Canada as well as in lesions reducing marketability of Yesso scallops in Japan. The impact of this bacterium on the health of mussels needs to be investigated in future research, especially since the cause of high mussel mortalities that have been occurring in France for the past few years is still undetermined.

Experimental infection of Mytilus edulis by two Vibrio splendidus-related strains: Determination of pathogenicity level of strains and influence of the origin and annual cycle of mussels on their sensitivity.

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.

High mortality of mussels in northern Brittany - Evaluation of the involvement of pathogens, pathological conditions and pollutants.

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.

A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe.

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.

Exploring First Interactions Between Ostreid Herpesvirus 1 (OsHV-1) and Its Host, Crassostrea gigas: Effects of Specific Antiviral Antibodies and Dextran Sulfate.

Viral entry mechanisms of herpesviruses constitute a highly complex process which implicates several viral glycoproteins and different receptors on the host cell surfaces. This initial infection stage was currently undescribed for Ostreid herpes virus 1 (OsHV-1), a herpesvirus infecting bivalves including the Pacific oyster, Crassostrea gigas. To identify OsHV-1 glyproteins implicated in the attachment of the virus to oyster cells, three viral putative membrane proteins, encoded by ORF 25, 41, and 72, were selected and polyclonal antibodies against these targets were used to explore first interactions between the virus and host cells. In addition, effects of dextran sulfate, a negative charged sulfated polysaccharide, were investigated on OsHV-1 infection. Effects of antiviral antibodies and dextran sulfate were evaluated by combining viral DNA and RNA detection in spat (in vivo trials) and in oyster hemolymph (in vitro trials). Results showed that viral protein encoded by ORF 25 appeared to be involved in interaction between OsHV-1 and host cells even if other proteins are likely implicated, such as proteins encoded by ORF 72 and ORF 41. Dextran sulfate at 30 µg/mL significantly reduced the spat mortality rate in the experimental conditions. Taken together, these results contribute to better understanding the pathogenesis of the viral infection, especially during the first stage of OsHV-1 infection, and open the way toward new approaches to control OsHV-1 infection in confined facilities.

A novel divergent group of Ostreid herpesvirus 1 µVar variants associated with a mortality event in Pacific oyster spat in Normandy (France) in 2016.

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.

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

Simultaneous detection of the protozoan parasites Toxoplasma, Cryptosporidium and Giardia in food matrices and their persistence on basil leaves.

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 = 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD = 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 <1 to 3 (oo)cysts/g and RR between 2 and 35%. This procedure was coupled to RT-qPCR analyses and showed that the three protozoa persisted on the leaves of basil and remained viable following storage at 4 °C for 8 days. These data strengthen the need to consider these protozoa in food safety.

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas.

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.

Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas.

The ostreid herpesvirus 1 (OsHV-1) and variants were implicated in mass mortality affecting the young Pacific cupped oysters, Crassostrea gigas, in European countries and those around the world. From 2008 onwards, oyster mortality had greatly increased on the French coast and was associated with the detection of a new OsHV-1 variant, entitled OsHV-1 µVar. The OsHV-1 µVar is predominant in oysters; however, other OsHV-1 variants have been detected in samples collected during mortality periods or collected out of mortality periods in France, Ireland, Spain, Portugal, Italy, Mexico, United States, South Korea, Australia, and New Zealand. A retrospective study conducted on 1047 OsHV-1 specimens sampled mainly in France between 2009 and 2012, revealed 17 undescribed OsHV-1 variants found in 65 oyster samples. These specimens presented point mutations situated downstream and upstream from the microsatellite area in the C region (ORF 4/5) which were different from the OsHV-1 reference and the OsHV-1 µVar. In the present work, investigation was performed to further characterize these OsHV-1 specimens by sequencing two habitually targeted regions to study genetic polymorphism of the virus: ORF 41/42 and ORF 35-38. An OsHV-1 variant detected in six oyster samples, contained a nucleotide substitution in the C region which impacted the amino acid sequence and might modify the function of the unknown protein encoding by ORF 4. For the ORF 41/42 region, only two specimens presented a synonymous mutation in comparison with the OsHV-1 µVar. All specimens contained the same deletion with the OsHV-1 µVar in ORF 35-38. Then, a phylogenetic analysis based on the C region was performed to investigate the distribution of undescribed specimens among 21 OsHV-1 DNA sequences notified in GenBank and collected from different countries (France, Japan, New Zealand, China, Ireland, and United States) between 1995 and 2012. All analyzed samples and the OsHV-1 µVar were placed in the same group, excepted for a Japan specimen. Our results contribute to improve the description of the genetic diversity of the OsHV-1 and the C region (ORF 4/5) appears to be a better target than ORF 42/42 and 35-38 to distinguish variants between themselves.

Genome exploration of six variants of the Ostreid Herpesvirus 1 and characterization of large deletion in OsHV-1µVar specimens.

The genetic polymorphism of the Ostreid Herpesvirus 1 (OsHV-1) has generally been investigated in three areas: ORFs 4/5, ORFs 42/43, and ORFs 35 to 38. The present study, however, focuses on 40 ORFs, representing 30% of the OsHV-1 genome, encoding four categories of putative proteins: 4 ORFs encoding putative inhibitor of apoptosis proteins; 17 ORFs encoding membrane proteins; 10 ORFs encoding secreted proteins; and 9 ORFs encoding RING finger proteins. The potential role of these proteins in major steps of the life cycle of the OsHV-1 motivated their selection. Seven specimens have been selected in accordance with their nucleotide variations in the C region (area located between the end of the ORF4 and the beginning of ORF 5): 3 OsHV-1µVar specimens, 2 OsHV-1µVar Δ9, one specimen of OsHV-1µVar Δ15, and one OsHV-1 specimen (reference control) close to the reference genome to validate PCRs. The OsHV-1µVar is mainly characterized by a deletion of 12 consecutive nucleotides followed by a deletion of one adenine in a microsatellite area located in the C region. A representation of nucleotide modifications between the different specimens was performed by building evolutionary trees with respect to the category of ORFs. This phylogenetic analysis revealed two groups: the first one corresponded to the reference control and the reference genome AY509253, and the second one included the 6 OsHV-1 variants. These results suggest that the two main groups come from the same common ancestor, and that the divergence between the reference OsHV-1 and its variants occurred quite far back in time. Moreover, consequences of nucleotide variations in the amino acid sequences, especially the change of the N glycoslyation sites, were investigated. Herein is the first report of four important deletions in these OsHV-1µVar variants: a deletion of 1385bp in ORF 11; a deletion of 599bp in ORF 48; a deletion of 3549bp in ORFs 61 to 64; and a deletion of 712bp in ORF 114. The size of the deletions differed between OsHV-1µVar specimens, OsHV-1µVar Δ9 specimens, and the OsHV-1µVar Δ15 specimen. These zones seem to correspond to special points of gene rearrangements for producing new proteins. Further investigation necessary proves to link such nucleotide modifications with consequences of protein functions in the OsHV-1 life cycle.

Physiological change under OsHV-1 contamination in Pacific oyster Crassostrea gigas through massive mortality events on fields.

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.

Proteomic expression of microfungal ripening starter Geotrichum candidum submitted to cold stress is strain-dependent: studies using 2d-dige technology and samespots software analysis.

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.

DNA adduct variations in non-smoking crop farmers: potential relationship with occupational exposure to pesticides?

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.