Adsorption of norovirus and ostreid herpesvirus type 1 to polymer membranes for the development of passive samplers.

AIMS: This study was performed to develop a passive sampling methodology for the detection of two viruses in seawater in the area of shellfish production, the norovirus (NoV), a human pathogen implicated in gastroenteritis outbreaks linked to oyster consumption and the ostreid herpesvirus type 1 (OsHV-1), a virus associated with mass mortalities of Pacific oysters. METHODS AND RESULTS: Commercially, membranes were tested for their capacity to adsorb virus: zetapor, gauze, nylon, low-density polyethylene (LDPE) and polyvinylidene difluoride (PVDF). Laboratory exposures of membranes to contaminated water samples (stool, sewage, seawater) were performed. Our data show that the amount of NoV GII genome per membrane measured with qRT-PCR increased with the time of exposure up to 24 h, for all types of membranes except gauze. After 15 days of exposure, the amount of NoV GII per membrane continued to increase only for nylon and LDPE. The amount of OsHV-1 per zetapor membrane was significantly increased as soon as 4 h of exposure, and after 24 h of exposure for all types of membranes. Exposure of membranes to serial dilutions of various samples revealed that the amount of NoV GII and OsHV-1 per membrane is significantly higher in diluted samples. The detection of NoV and OsHV-1, respectively, with zetapor and PVDF membranes was found to be more efficient than the direct analysis of sewage and seawater. CONCLUSIONS: All membranes immersed in contaminated samples adsorbed NoV GII and OsHV-1. The amount of both viruses increased with the time of exposure. Zetapor and PVDF membranes seem to be more adapted to NoV GII and OsHV-1 detection respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Membranes tested will be used as passive samplers to improve the detection of virus in oyster production areas. Also, passive samplers could be a valuable tool for microbiome analysis with new generation sequencing.

Immunotoxicological effects of environmental contaminants on marine bivalves.

Coastal areas are complex environments frequently contaminated by numerous pollutants that represent a potential threat to marine organisms, especially bivalves. These pollutants may have major ecological consequences. Although effects of different environmental contaminants on the immune system in marine bivalves have been already reported, a few of reviews summarizes these effects. The main purpose of this chapter relies on summarizing recent body of data on immunotoxicity in bivalves subjected to contaminants. Immune effects of heavy metals, pesticides, HAP, PCB and pharmaceuticals are presented and discussed and a particular section is devoted to nanoparticle effects. A large body of literature is now available on this topic. Finally, the urgent need of a better understanding of complex interactions between contaminants, marine bivalves and infectious diseases is noticed.

A European epidemiological survey of Vibrio splendidus clade shows unexplored diversity and massive exchange of virulence factors.

The Vibrio splendidus clade has previously been associated with epidemic outbreaks of various aquatic animals, as in the case of the cupped oyster, Crassostrea gigas. To investigate whether involved strains could present a clonal origin and to identify possible alternative background carriage animals or zooplankton, a large epidemiological survey was conducted on isolates of the splendidus clade. For this purpose, Vibrio strains were isolated from various samples including oysters, mussels, sediments, zooplankton, and sea water on the basis of a North/South gradient of the European sea water zone (Ireland, The Netherlands, France, Italy, and Spain). A total of 435 isolates were successfully associated to the V. splendidus clade using real time polymerase chain reaction with 16S specific primers and probes. A multiple-locus variable-number tandem-repeat analysis (VNTR) was conducted on all isolates based on a multiplex PCR-VNTR with a set of primer pairs designed from the V. tasmaniensis LGP32 genome. Preliminary validation of the primers on a set of collection strains from the V. splendidus clade confirmed that the former V. splendidus-related LGP32 and relative strains were related to V. tasmaniensis rather than to the type strain V. splendidus LMG 4042. The VNTR analysis was then successfully conducted on 335 isolates which led to the characterization of 87 different profiles. Our results showed that (1) the high diversity of VNTR did not enlighten significant correlation between a specific pattern and the origin of collected samples. However, populations isolated from animal samples tend to differ from those of the background environment; (2) oyster mortality events could not be linked to the clonal proliferation of a particular VNTR type. However, few different patterns seemed successively associated with samples collected during peaks of oyster's mortality. (3) Finally, no correlation could be seen between specific VNTR patterns and sequence phylogeny of the virulence factors vsm and ompU that were detected among strains isolated during as well as outside mortality events. These results, combined with incongruence observed between the ompU and vsm phylogenetic trees, suggested both large diffusion of strains and massive lateral gene transfer within the V. splendidus clade.

Effects of low-dose exposure to pesticide mixture on physiological responses of the Pacific oyster, Crassostrea gigas.

This study investigated the effects on the physiology of Pacific oyster, Crassostrea gigas, of a mixture of pesticides containing 0.8 µg L(-1) alachlor, 0.6 µg L(-1) metolachlor, 0.7 µg L(-1) atrazine, 0.6 µg L(-1) terbuthylazine, 0.5 µg L(-1) diuron, 0.6 µg L(-1) fosetyl aluminum, 0.05 µg L(-1) carbaryl, and 0.7 µg L(-1) glyphosate for a total concentration of 4.55 µg L(-1) . The total nominal concentration of pesticides mixture corresponds to the pesticide concentrations in the shellfish culture area of the Marennes-Oleron basin. Two varieties of C. gigas were selected on the foreshore, based on their characteristics in terms of resistance to summer mortality, to assess the effects of the pesticide mixture after 7 days of exposure under controlled conditions. The early effects of the mixture were assessed using enzyme biomarkers of nitrogen metabolism (GS, glutamine synthetase), detoxification metabolism (GST, glutathione S-transferase), and oxidative stress (CAT, catalase). Sublethal effects on hemocyte parameters (phagocytosis and esterase activity) and DNA damages (DNA adducts) were also measured. Changes in metabolic activities were characterized by increases in GS, GST, and CAT levels on the first day of exposure for the "resistant" oysters and after 3-7 days of exposure for the "susceptible" oysters. The formation of DNA adducts was detected after 7 days of exposure. The percentage of hemocyte esterase-positive cells was reduced in the resistant oysters, as was the hemocyte phagocytic capacity in both oyster varieties after 7 days of exposure to the pesticide mixture. This study highlights the need to consider the low doses and the mixture of pesticides to evaluate the effects of these molecules on organisms.

New insights in flat oyster Ostrea edulis resistance against the parasite Bonamia ostreae.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality in flat oyster stocks in Europe. Control of the disease currently relies on disease management practices and transfer restriction. Previously, massal selections based on survival to challenge to infection with B. ostreae have been applied to produce flat oyster families with resistant progeny. In an attempt to understand the molecular mechanisms involved in disease resistance, differentially expressed sequence tags between resistant and wild Ostrea edulis haemocytes, both infected with the parasite, were identified using suppression subtractive hybridisation. Expression of seven ESTs has been studied using quantitative reverse-transcriptase PCR. The base-line expression of an extracellular superoxide dismutase, inhibitor of apoptosis (OeIAP), Fas ligand (OeFas-ligand) and Cathepsin B was significantly increased, whilst cyclophilin B appeared significantly decreased in resistant oysters. Considering their great interest for further studies, the open reading frames of the OeFas-ligand and OeIAP were completely sequenced.

Detection of Bonamia ostreae and B. exitiosa (Haplosporidia) in Ostrea edulis from the Adriatic Sea (Italy).

The flat oyster Ostrea edulis L. is widespread along the Italian coasts. In particular, the Manfredonia Gulf (Adriatic Sea) represents an important site where natural beds subsist. Previous monitoring conducted in 1990 by light microscopy and ultrastructural studies revealed the presence of Bonamia-like microcell parasites in some flat oysters: following this observation, a new sampling of O. edulis was carried out at this location in 2007. Of 750 oysters collected, 3 showed the presence of uninucleated microcells (2 to 3 microm diameter) free or inside the haemocyte cytoplasm by cytology and histopathology. Molecular analysis confirmed that the microcells in 2 oysters were B. exitiosa, whereas in the third oyster the microcells were B. ostreae. Moreover, molecular studies were carried out to confirm the existence of Bonamia sp. in archived samples, confirming the presence of B. ostreae in the Manfredonia Gulf since 1990.

Infestation of the cupped oysters Crassostrea angulata, C. gigas and their first-generation hybrids by the copepod Myicola ostreae: differences in susceptibility and host response.

SUMMARY: We studied the prevalence and intensity of the parasitic copepod Myicola ostreae in 2 closely related oysters Crassostrea angulata and C. gigas and their F1 hybrids. The effects on host and host reaction were also analysed to better understand host-parasite relationships between copepods and bivalve molluscs. Full reciprocal crosses were carried out between C. angulata and C. gigas and the progenies were reared in the wild in Ria Formosa Lagoon (Portugal), allowing natural infestation by M. ostreae. Prevalence and intensity were significantly higher in C. angulata than in C. gigas. The parasite level of F1 hybrids was similar to C. angulata and significantly higher than in C. gigas. The results of our study support a hypothesis of dominantly inherited susceptibility to M. ostreae infestation. Moreover, copepods were observed on the gill surface of C. gigas engulfed by a capsule-like structure. Histological analyses revealed that the copepods were surrounded by a massive agglomerate of haemocyte-like cells encircled by a thin layer of fibroblast-like cells. This encapsulation response was not observed in C. angulata or in F1 hybrids. These results suggest that the differential susceptibility to M. ostreae between C. angulata and C. gigas may be ascribed to host defence factors.

Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR.

Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.

Recent findings on phenoloxidases in bivalves.

The production of melanin is a complex process involving biochemical cascades, such as the pro-phenoloxidase (proPO) system, and enzymes, such as phenoloxidases (POs). Different studies have shown a strong correlation between the decrease in PO activities and the occurrence of diseases in bivalve invertebrates, leading to mortalities in the host. Results of these studies suggest that POs could play a fundamental role in defense mechanisms in bivalves. This article reviews the fundamental knowledge on the proPO system in bivalves and the methods used to assess PO activities. Finally, this is the first report on the major findings of laboratory and field studies that indicate that a type of PO in bivalves, the laccase enzyme, is inducible and involved in the 1) immune 2) antioxidant and 3) detoxification roles in bivalves, and might be an ecological potential biomarker of environmental stress.

[Viruses of bivalve shellfish]. / Les virus infectant les bivalves marins.

Bivalve molluscs are filter feeders and as a consequence they may bioaccumulate in their tissues viruses that infect humans and higher vertebrates. However, there have also been described mortalities of bivalve molluscs associated with viruses belonging to different families. Mass mortalities of adult Portuguese oysters, Crassostrea angulata, among French livestocks (between 1967 et 1973) were associated with irido-like virus infections. Herpesviruses were reported in the eastern oyster, Pacific oyster and European flat oyster and lately in scallops and clams. Disseminated neoplasia, a proliferative cell disorder of the circulatory system of bivalves, although of uncertain aetiology, has been suggested to be caused by retroviral infections. Other viruses described in bivalves are interpreted as members of the Papovaviridae, Togaviridae, Reoviridae, Birnaviridae and Picornaviridae. However, the lack of bivalve cell lines renders difficult virus isolation from molluscs.

Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas).

Ostreid Herpesvirus 1 (OsHV-1) was likely detected in Pacific oysters, Crassostrea gigas, at different stages of development. Viral infections were associated with high mortality rates in the spat and larvae. Furthermore, the persistance of OsHV-1 in asymptomatic adults was demonstrated by detection of viral DNA and proteins. In the present study, three successive generations of C. gigas (G0 and G1 parental oysters, G1 and G2 larvae) were screened for OsHV-1 by PCR. Viral DNA was detected in 2-day-old larvae, indicating that infection may take place at very early stages. Although results strengthen the hypothesis of a vertical transmission, it was not possible to predict the issue of a particular type of cross. Indeed, the detection of viral DNA in parental oysters did not systematically correspond to a productive infection or result in a successful transmission to the progeny. However, the infective status of the parents appeared to have an influence on both the infection and the survival rates of the progeny. Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of cross. These results suggest that OsHV-1-infected females may transmit to their offspring some kind of protection or resistance against viral infection.

Survey for neoplasia in Macoma balthica from the Gulf of Gdansk by flow cytometry.

Using flow cytometry, 234 Macoma balthica were examined during a survey to determine frequency of neoplasia in the Gulf of Gdansk (Poland). Clams were collected in 4 locations and DNA content in gill tissue cells was determined by flow cytometry using propidium iodide staining. Cell permeabilization was induced by osmotic shock. Prevalence of neoplasia ranged from 9.6 to 26.7% depending on location. DNA content in aneuploid cells was higher than in normal dividing cells. The fluorescence value for aneuploid cells corresponded to tetraploid/pentaploid cells. Three stages of neoplasia were defined, based on the percentage of aneuploid cells determined by flow cytometry. Histopathological and cytogenetic analyses were also carried out on the same clams for comparative study. Proportions of normal and affected clams detected using flow cytometry were similar to those identified using both methods. In the present study, no clear relationship was demonstrated between prevalence of neoplasia and pollutant detection in the different sampling sites.

Neoplasia detection in Macoma balthica from the Gulf of Gdansk: comparison of flow cytometry, histology and chromosome analysis.

A flow cytometry protocol was applied for the detection of neoplasia in Macoma balthica L. from the Gulf of Gdansk (Baltic Sea, Poland). A simple method, based on an osmotic shock, was used to permeabilise gill cells. The cytometric pattern of normal clams consisted of 2 peaks, a major peak B and a smaller peak C. The cytometric pattern of affected clams consisted of 2 peaks named B' and C'. Two parameters were used to define the stages of abnormalities in M. balthica clams based on the percentage of cells in peaks B, C, B' and C' and on the ratio between the fluorescence value of peaks B, C, B' and C' in all individuals. Three stages of neoplasia were clearly distinguished by flow cytometry considering peak C'. Stage 1 was characterised by a major population of cells in peak B' and more than 10% of cells in the C' peak. Stage 2 consisted of a lower percentage of cells in peak B' and more than 25% of cells in peak C'. Stage 3 of the neoplasia was characterised by a further reduction in peak B' and more than 40% of cells in peak C'. Flow cytometry allowed for objective detection of neoplasia and provided a rapid method for measuring the DNA content of thousands of cells per individual. The accuracy of flow cytometry was assessed by comparing with standard histological techniques, used here as a reference technique for the detection of neoplasia, and with chromosome analysis. All individuals were analysed in parallel using the 3 techniques. The proportion of normal and affected individuals diagnosed using flow cytometry was comparable to the proportion determined by histology and chromosome analysis.

Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes.

Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.

Monoclonal antibodies to European flat oyster Ostrea edulis hemocytes: characterization and tissue distribution of granulocytes in adult and developing animals.

Monoclonal antibodies specific for hemocyte sub-populations of the European flat oyster, Ostrea edulis, were prepared using separated granulocytes and hyalinocytes as antigen sources. Six monoclonal antibodies specific for hemocytes were selected. Five of them showed a specificity for more than one hemocyte type and one hybridoma produced a monoclonal antibody reacting specifically with granulocytes. At the ultrastructural level, this monoclonal antibody demonstrated epitopes principally in dense cytoplasmic granules of granulocytes. Western blotting analysis indicated that a peptide of 50kDa was recognized by this antibody. It was therefore used to investigate granulocyte distribution and ontogenesis in European flat oysters using immunohistochemistry. Granulocytes were mostly observed in connective tissues in different organs. Their distribution pattern in digestive gland, mantle, gills and gonad may indicate different functional status. Moreover, the absence of granulocytes in early larval stages can partly correspond to an immature immune system in oyster larvae.

Herpes-like virus detection in infected Crassostrea gigas spat using DIG-labelled probes.

An in situ hybridization protocol for detecting the herpes-like virus which infects French Pacific oysters, Crassostrea gigas, was developed. Two DNA probes were synthesized by incorporation of digoxigenin 11-dUTP during PCR. Two oyster herpes-like virus specific primer pairs, A5/A6 and C1/C6, were used. Both DIG-labelled probes were able to detect 50 pg of herpes-like virus PCR amplified DNA in Southern blot hybridizations. The probes hybridized with viral DNA in paraffin sections of infected C. gigas spat. No non-specific binding was observed. The ability of the defined in situ hybridization technique to diagnose herpes-like virus infections in oysters was compared with light and transmission electron microscopy techniques in infected and non-infected spat. In situ hybridization assays were also conducted on paraffin sections to determine virus distribution within the host and to study the pathogenesis infection. In situ hybridization confirmed that the expression pattern of the herpes-like virus was restricted to connective tissues as described previously by light and transmission electron microscopy. However, this technique also allowed the detection of viral DNA in the oyster nervous system. Some labelled cells were observed in the visceral ganglion of infected oyster spat.

Development and use of an internal standard for oyster herpesvirus 1 detection by PCR.

Oyster samples were examined using a competitive PCR method in order to detect and quantify oyster herpesvirus 1 (OsHV-1) DNA. Quantitation of viral DNA by competitive PCR assay was based on co-amplification of OsHV-1 DNA and a competitor where a known amount of competitor DNA was present in the same reaction mixture. The competitor was engineered so that it differs in length (deletion of 76 base pairs) from the viral DNA. The assay allowed the detection of 1 fg of viral DNA among 0.5 mg of oyster tissues. The method was used to demonstrate the absence of PCR inhibitors in oyster spat ground tissues. PCR inhibition was observed in adult oyster samples when the same tissue preparation procedure was used. On the contrary, classical phenol/chloroform DNA extraction from adult oyster tissues allowed co-amplification of the internal standard competitor and the viral DNA. The method was successfully used to demonstrate the presence of viral DNA in asymptomatic adult oysters. Quantitation of OsHV-1 DNA in infected spat and asymptomatic adult oysters was also carried out. Viral DNA (1.5-325 pg) were detectable in 0.5 mg of oyster tissues in adults. The amounts of viral DNA in infected oyster spat varied from 750 pg to 35 ng per 0.5 mg of ground tissues.

Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France.

A PCR-based procedure for detecting a herpes-like virus that infects the Japanese oyster, Crassostrea gigas, in France was developed. Two primers were designed to provide specific amplification products ranging in size from 917 to 1001 bp when carried out on oyster herpes-like virus DNA. No amplification was observed of oyster genomic DNA nor of the DNA from vertebrate herpesviruses. Crude samples were prepared and submitted to nested PCR, allowing amplification of DNA fragments of the expected size when carried out on infected larval and spat samples. The procedure used to prepare the sample for PCR was found to be critical because of the presence of unidentified substances in oyster tissues that inhibit the PCR reaction. A rapid and convenient sample preparation using ground tissues allowed a sensitive detection of the herpes-like virus infected oysters. The ability of the defined PCR protocol to diagnose herpes-like virus infections in oysters was compared to the transmission electron microscopy technique using 15 C. gigas larval batches with or without mortalities. PCR amplification is as sensitive a diagnostic assay for herpes-like virus as transmission electron microscopy. However, the nested PCR protocol is more convenient and less time consuming. The relationship between reported mortalities among C. gigas oyster spat and herpes-like virus DNA detection by PCR was also investigated. Statistical analysis showed that virus detection and mortalities are correlated. This observation highlights the importance of studying the causative role of herpes-like virus in oyster spat mortalities.

Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation.

In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200bp) were designed (C(9)/C(10) and B(4)/B(3)). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C(9)/C(10), which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data.

A herpes-like virus infects a non-ostreid bivalve species: virus replication in Ruditapes philippinarum larvae.

Sporadic high mortalities were reported in June 1997 among hatchery-reared larval Manila clam Ruditapes philippinarum in a French commercial hatchery. Cellular abnormalities were observed in semi-thin sections in affected animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in larvae. This is the first description of a herpes-like virus infection in larval R. philippinarum, a non-ostreid bivalve species. Virus particles were similar to other herpes-like viruses described from different oyster species with respect to ultrastructure and morphogenesis. Electron microscopic examination also demonstrated cells with condensed chromatin and extensive perinuclear fragmentation of chromatin. Like viruses infecting oysters, the herpes-like virus detected in clams may induce apoptosis in infected animals.