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ABSTRACT: In this study, we described the cytosolic HSP90 of Bonamia ostreae, an intracellular parasite of Ostrea edulis hemocytes. The complete open reading frame was assembled by Rapid Amplification cDNA Ends reactions on cDNA of B. ostreae-infected hemocytes. HSP90 amplification was corroborated in infected oysters and B. ostreae purified cells. The functionality of the HSP90, studied by inhibitory assays with radicicol, suggests that this protein may play a role in hemocyte invasion. Our results inform the molecular basis that governs B. ostreae-O. edulis interactions.
Journal of Eukaryotic Microbiology 03/2013; · 2.66 Impact Factor
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ABSTRACT: Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. The life cycle of this species is still poorly known, although there is evidence of the need for intermediate host(s). In the present study, we have used molecular approaches to identify this parasite in samples of the dwarf oyster Ostrea stentina after reports of massive mortality along the Tunisian coasts. In 2009 we evaluated the status of O. stentina from Monastir and checked if there was an infection with M. refringens, using polymerase chain reaction assays. Of the 103 tested O. stentina, 85 were PCR-positive using a Marteilia genus-specific assay. Additional assays were subsequently carried out on some samples collected in 2010 in Monastir and processed for histology, transmission electron microscopy and complementary molecular analyses. PCR was carried out to amplify the IGS and ITS regions. Histological and transmission electron microscopy analyses allowed us to confirm the presence of this parasite in the digestive gland tissue of O. stentina and to characterize it at the ultrastructural level. This is the first record of the occurrence of M. refringens in the oyster O. stentina along the Tunisian coasts.
Journal of Invertebrate Pathology 12/2012; · 2.06 Impact Factor
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ABSTRACT: Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C. gigas. On December 2003, naïve and Bonamia-relatively resistant flat oysters from Ireland, Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O(2)(-)] and hydrogen peroxide [H(2)O(2)]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreaein vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded.
Journal of Invertebrate Pathology 03/2012; 109(3):274-86. · 2.06 Impact Factor
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Epidémiologie et Santé animale. 01/2012; 62:27-42.
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Céline Garcia,
Anne Thébault,
Lionel Dégremont, Isabelle Arzul,
Laurence Miossec,
Maeva Robert,
Bruno Chollet,
Cyrille François,
Jean-Pierre Joly,
Sylvie Ferrand,
Nolwenn Kerdudou,
Tristan Renault
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ABSTRACT: ABSTRACT: Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.
Veterinary Research 06/2011; 42(1):73. · 4.06 Impact Factor
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ABSTRACT: Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite, B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite, B. ostreae.
International journal for parasitology 03/2011; 41(7):755-64. · 3.39 Impact Factor
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Isabelle Arzul,
Aimé Langlade,
Bruno Chollet,
Maeva Robert,
Sylvie Ferrand,
Emmanuelle Omnes,
Sophie Lerond,
Yann Couraleau,
Jean-Pierre Joly,
Cyrille François,
Céline Garcia
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ABSTRACT: Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It can be detected in juveniles but mortalities mainly affect oysters which are more than 2 years old. The parasite is usually observed inside haemocytes and sometimes free, notably in gill epithelia suggesting a parasite release through this organ. However, the infective form and ways of entry and release remain undetermined. Flat oysters incubate their larvae in their pallial cavity for 8-10 days before releasing them into the water column. Flat oysters in Bay of Quiberon in South Brittany (France) are known to be infected with B. ostreae since 1979 and is the most important area in France for O. edulis spat collection. Flat oysters incubating larvae were sampled in this area during summertime between 2007 and 2009. Both adults and larvae were preserved and assayed by PCR and in situ hybridisation (ISH). PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific labelling could be detected by ISH in gills, digestive system, gonad and mantle in adults and in the epithelium surrounding the visceral cavity of some larvae. Our results demonstrate that larvae can be infected with B. ostreae. Larvae might thus contribute to the spread of the parasite during their planktonic life. In addition, their transfer for aquaculture purpose should be controlled especially when they are exported from infected zones.
Veterinary Parasitology 02/2011; 179(1-3):69-76. · 2.58 Impact Factor
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ABSTRACT: Bonamia ostreae is an intrahaemocytic protozoan affecting Ostrea edulis. The parasite multiplies within haemocytes without being degraded and involves changes in cellular activities. Studies aiming at better understanding host response to a pathogen at the transcriptome levels are frequently based on the use of real time PCR assays, which require some reference genes. However, very few sequence data is available for O. edulis in public databases. Subtracted cDNA libraries were constructed from the O. edulis haemocytes in order to identify genes involved in host reactions against the parasite and quantitative real time PCR assays were developed to study expression of these genes. In this context, identification of reference genes and study of their relative expression stability were required for quantitative real time PCR normalization. The expression of 5 potential candidate reference genes from O. edulis (ie elongation factor 1 alpha (EF1-α), 60S ribosomal protein L5 (L5), glyceraldehyde 3-phosphate-dehydrogenase (GAPDH), polyubiquitin (Ubiq) and β-actin (ACT)) was studied using RNAs extracted from pools of haemocytes in contact with the parasite B. ostreae and haemocytes alone. Gene expression was quantified by real time PCR and expression stability was analysed with two analytical approaches GeNorm and NormFinder. GAPDH and EF1-α were identified as the most stable genes with the GeNorm analysis. Whatever were the tested conditions, EF1-α was also found as the most stable gene using Normfinder. The less stable gene was β-actin although this gene is commonly used as housekeeping gene in many studies. Our results suggest using GAPDH and EF1-α combined as reference genes when studying expression levels in haemocytes of O. edulis. In addition, the complete ORF of these two genes was characterized.
Fish & Shellfish Immunology 12/2010; 29(6):937-45. · 3.32 Impact Factor
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ABSTRACT: Bonamiosis due to the parasite Bonamia ostreae is a disease affecting the flat oyster Ostrea edulis. B. ostreae is a protozoan, affiliated to the order of haplosporidia and to the cercozoan phylum. This parasite is mainly intracellular, infecting haemocytes, cells notably involved in oyster defence mechanisms. Suppression subtractive hybridisation (SSH) was carried out in order to identify oyster genes differentially expressed during an infection of haemocytes with B. ostreae. Forward and reverse banks allowed obtaining 1104 and 1344 clones respectively, among which 391 and 480 clones showed a differential expression between both tested conditions (haemocytes alone versus haemocytes in contact with parasites). ESTs of interest including genes involved in cytoskeleton, respiratory chain, detoxification membrane receptors, and immune system were identified. The open reading frames of two selected genes (galectin and IRF-like) were completely sequenced and characterized. Real time PCR assays were developed to study the relative expression of candidate ESTs during an in vitro infection of haemocytes by live and dead parasites. Haemocyte infection with B. ostreae induced an increased expression of omega glutathione S-transferase (OGST), superoxide dismutase (SOD), tissue inhibitor of metalloproteinase (TIMP), galectin, interferon regulatory factor (IRF-like) and filamin genes.
Developmental and comparative immunology 11/2010; 35(3):323-33. · 3.29 Impact Factor
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ABSTRACT: Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of the Pacific oyster (Crassostrea gigas). In the summer 2008, abnormal mortality rates ranging from 80% to 100% were reported in France and affected only Pacific oysters. Analyses of oyster samples collected during mortality outbreaks demonstrated a significant detection of OsHV-1 (75% of analysed batches), which appeared stronger than previous years. DNA sequencing based on C and IA regions was carried out on 28 batches of OsHV-1 infected Pacific oysters collected in 2008. Polymorphisms were described in both the C and IA regions and characterized a genotype of OsHV-1 not already reported and termed OsHV-1 microVar. A microsatellite zone present in the C region showed several deletions. Additionally, 44 isolates collected in France and in the USA, from 1995 to 2007 were sequenced and compared to the 2008 sequences. The analyses of 76 sequences showed OsHV-1 microVar detection only in 2008 isolates. These data suggest that OsHV-1 microVar can be assumed as an emergent genotype.
Virus Research 10/2010; 153(1):92-9. · 2.94 Impact Factor
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ABSTRACT: Bonamiosis due to the intrahaemocytic protistan parasite Bonamia ostreae is a European endemic disease affecting the flat oyster Ostrea edulis. The parasite has been described in various ecosystems from estuaries to open sea, but no clear correlation has yet been demonstrated between disease development and environmental parameters. In this study, the effect of temperature and salinity on the survival of purified parasites maintained in vitro in seawater was investigated by flow cytometry. Purified parasites were incubated in various seawater media (artificial seawater, natural seawater, seabed borewater) at various temperatures (4, 15 and 25 degrees C) and subjected to a range of salinities from 5 to 45 g l(-1). Parasites were collected after 12, 24 and 48 h of incubation for flow cytometry analyses including estimation of parasite mortality and parasite viability through detection of non-specific esterase activities. Artificial seawater appeared unsuitable for parasite survival, and results for all media showed a significantly lower survival at 25 degrees C compared to 4 degrees C and 15 degrees C. Moreover, high salinities (> or = 35 g l(-1)) favoured parasite survival and detection of esterase activities. Flow cytometry appears to be a suitable technique to investigate survival and activities of unicellular parasites like B. ostreae under varied conditions. Although these results contribute to a better understanding of existing interactions between the parasite B. ostreae and its environment, validation through epidemiological surveys in the field is also needed.
Diseases of Aquatic Organisms 05/2009; 85(1):67-75. · 2.20 Impact Factor
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ABSTRACT: Herpes-like viral infections have been reported in different bivalve mollusc species throughout the world. High mortalities among hatchery-reared larvae and juveniles of different bivalve species have been associated often with such infections. The diagnosis of herpes-like viruses in bivalve molluscs has been performed traditionally by light and transmission electron microscopy. The genome sequencing of one of these viruses, oyster herpesvirus 1 (OsHV-1), allowed the development of DNA-based diagnostic techniques. The polymerase chain reaction (PCR) has been used for the detection of OsHV-1 DNA in bivalve molluscs at different development stages. In addition, the PCR used for detection of OsHV-1 has also allowed the amplification of DNA from an OsHV-1 variant. The literature on DNA extraction methods, primers, PCR strategies, and confirmatory procedures used for the detection and identification of herpesviruses that infect bivalve molluscs are reviewed.
Journal of Virological Methods 02/2007; 139(1):1-11. · 2.01 Impact Factor
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ABSTRACT: The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study.
Diseases of Aquatic Organisms 08/2006; 71(1):75-80. · 2.20 Impact Factor
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ABSTRACT: An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.
Diseases of Aquatic Organisms 08/2006; 71(1):81-5. · 2.20 Impact Factor
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ABSTRACT: Viral gametocytic hypertrophy was reported for the first time in 2001 in Pacific oyster Crassostrea gigas in France. Since this date, the number of reported cases and the distribution area have increased every year; however, the cases are not associated with macroscopic signs or increased mortality rates. Both male and female gametes were hypertrophied and basophilic inclusions were observed in gamete nuclei. Transmission electron microscopy revealed the presence of viral particles in these intranuclear basophilic inclusions. These particles had characteristics similar to those of the Papillomaviridae and Polyoma viridae families: they were small, non-enveloped, icosahedral, and 44 to 56 nm in diameter. The viral particles were found in male, female and hermaphrodite oysters and no significant difference in viral infection was observed between those groups. The frequency of detection and the intensity of infection were low and no host defence reaction was recognised, suggesting that the viral particles had a weak impact on C. gigas. The viral particles described in the present study seem to be similar to these described in C. virginica in the USA and Canada and in C. gigas in Korea, but further studies are required to confirm their identity. The issue of a possible emergence of this infection is discussed.
Diseases of Aquatic Organisms 07/2006; 70(3):193-9. · 2.20 Impact Factor
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ABSTRACT: Since 1972, several herpes-like virus infections have been reported among different bivalve species around the world. Most of these reports involved larvae or juveniles presenting high mortalities. Two case reports of herpes-like viruses concerned adult oysters, Crassostrea virginica in USA and Ostrea angasi in Australia. Molecular techniques including PCR and in situ hybridization (ISH) have been recently developed to detect the oyster herpesvirus genome. In the present study, 30 Pacific oyster, Crassostrea gigas, adults have been analyzed using three different techniques: PCR, ISH and immunochemistry, in order to detect herpesviruses in asymptomatic individuals. PCR and ISH allowed detection of oyster herpesvirus DNA in 93.3 and 86.6%, respectively, of analyzed oysters while polyclonal antibodies allowed detection of viral proteins in 76.6% of analyzed adult oysters. These results suggest that oyster herpesvirus infects adult oysters with high prevalence and that the virus may persist in its host after primary infection. The detection of viral DNA and viral proteins in the gonad of several individuals supports the hypothesis of a possible vertical transmission of the infection. Lastly, concordance among the three techniques used in this study is discussed.
Virus Research 04/2002; 84(1-2):151-60. · 2.94 Impact Factor
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ABSTRACT: Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.
Molecular and Cellular Probes.
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Tristan Renault,
Gwenhael Allain, Isabelle Arzul,
Bruno Chollet,
Laetitia Cobret,
Sophie De Decker,
Nicole Faury,
Sylvie Ferrand,
Cyrille Francois,
Celine Garcia,
Philippe Haffner,
Jean-Pierre Joly,
Laurence Miossec,
Benjamin Morga,
Jean-Louis Nicolas,
Emmanuelle Omnes,
Jean-francois Pepin,
Denis Saulnier,
David Schikorski,
Amelie Segarra
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ABSTRACT: Abnormal mortality outbreaks occurred in the majority of Pacific oyster (Crassostrea gigas) producing areas in France in 2008 during the summer period. These outbreaks were usually sudden and severe (up to 100%) and affected essentially spat (oysters less than 1 year-old) and juveniles (12 to 18 month-old oysters). Crassostrea gigas was the single affected species Mortality outbreaks were first observed from mid May to mid June in Brittany, Vendée, Charente Maritime and Mediterranean coast A second episode of massive mortality events was reported at the end of June and the beginning of July. All oyster production sites were affected except some particular locations including Arcachon Bay and Brittany. Mortality occurred latter in Arcachon Bay in August. Different hypothesis including the implication of environmental factors, toxic algae or pathogens have been explored. In this context, samples were collected from all affected locations and tested in order to search pathogens. Different tests were carried out including histology, bacteriology, PCR for the detection of OsHV-1 and Vibrio splendidus and V. aestuarianus, transmission electron microscopy. Some experimental assays were also performed in order to reproduce mortality and to test the hypothesis of an infectious aetiology. Results of diagnostic tests indicated that (1) there was no listed pathogen involved, (2) OsHV-1 was detected in most of samples especially in moribund oysters, (3) Vibrio splendidus, V. aestuarianus and V. harveyi were detected in many samples and (4) viral particles looking like herpes viruses were observed by TEM in moribund tested oysters. Results of the experimental assays showed that mortalities were reproducible by injecting in the adductor muscle of healthy oysters tissue suspensions from oysters expressing mortalities. The suspensions filtrated at 0.22 µm induced mortalities whereas suspensions filtrated at 0.1 µm did not induce any mortality suggesting that these mortalities require an agent between 100 and 200 nm in size. Analysis of experimentally injected oysters revealed the presence of high amounts of OsHV-1 DNA by Q-PCR and TEM analysis demonstrated the presence of herpes virus particles in experimentally infected oysters. Present conclusions are that there is no listed pathogen and the reported mortalities are associated with bacterial and OsHV-1 infections. However, considering that these pathogens are usually detected in mortality events affecting C. gigas, a question has been arisen: why oyster mortalities were so massive in 2008? Further works are presently conducted in order to identify additional factors including the emergence of highly virulent strains and particular environmental conditions which might be necessary to fully explain the situation encountered in 2008.
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William G Dundon, Isabelle Arzul,
Emmanuelle Omnes,
Maeva Robert,
Cristian Magnabosco,
Michela Zambon,
Lorenzo Gennari,
Anna Toffan,
Calogero Terregino,
Ilaria Capua,
Giuseppe Arcangeli
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ABSTRACT: In April 2010, the sampling of juvenile Pacific cupped oysters originating from France was undertaken from a farm located off the coast of the Marche region in Italy. Samples were sent to the national reference laboratory for mollusc diseases for bacteriological, histological, electron microscopical and molecular analysis. Classical and Real-Time PCR indicated the presence of the microvariant (OsHV-1 μvar) of Type 1 Ostreid Herpes virus (OsHV-1) despite the absence of clinical and pathological signs normally associated with the presence of this variant. Further molecular and sequencing analysis revealed the presence of OsHV-1 μvar and OsHV-1 DNA in one oyster indicating that simultaneous detection of both viruses is possible. This is the first report of the presence of OsHV-1 μvar in Italy and also the first time that evidence of possible co-infection of OsHV-1 and OsHV-1 μvar has been provided.
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ABSTRACT: Sporadic high mortalities were reported among larval French scallops (Pecten maximus). Electron microscopy of moribund larvae revealed particles with the characteristics of a herpesvirus in association with cellular lesions. PCR and DNA sequencing showed that the virus is a variant of ostreid herpesvirus-1 that has already been described in clams and oysters. This is the first description of a herpesvirus infection of a scallop species. The virus was transmitted successfully from an extract of infected scallop larvae to uninfected scallop or oyster (Crassostrea gigas) larvae, demonstrating that it is able to infect both species. Detection of viral DNA in asymptomatic adult scallops by in situ hybridisation indicates that the herpesvirus may have been transmitted from adults to larvae. It is notable that, unlike most herpesviruses, this virus has a wide host range reflected by its ability to infect several species of marine bivalve.
Virology.
