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.

Concomitant sporadic high mortalities were reported in France in May 1994 among batches of hatchery-reared larval Pacific oysters Crassostrea gigas and European flat oysters Ostrea edulis in 2 hatcheries, and in June and July 1994 among batches of cultured spat of both species in a shellfish nursery. Histological observation showed the presence of cellular abnormalities in moribund animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in infected larvae and spat of both oyster species. This is the first description of a herpes-like virus infection in larval O. edulis. Viruses observed in diseased larvae and spat of both species are similar with respect to ultrastructure and morphogenesis. They were detected simultaneously in C. gigas and O. edulis larvae and spat, indicating possible interspecific transmission. Moreover, these viruses are associated with high mortality rates in both oyster species. An electron microscopic examination revealed hemocytes with condensed chromatin and extensive perinuclear fragmentation of chromatin. These data suggest that herpes-like viruses infecting oysters may induce apoptosis in oyster hemocytes. KEY WORDS: Herpes-like virus · European flat oyster · Ostrea edulis · Pacific oyster · Crassostrea gigas · Oyster mortality · Concomitant infection · Virus replication · Apoptosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 42: [173][174][175][176][177][178][179][180][181][182][183] 2000 viral particles reported by Hine et al. (1992) in New Zealand and the virus-like particles described by Comps & Cochennec (1993) among French cultured European flat oyster spat.We describe for the first time a herpes-like virus infecting European flat oyster larvae and concomitant herpes-like virus infections associated with high mortalities among hatchery-reared larvae and nurserycultured spat of both Crassostrea gigas and Ostrea edulis during the summer of 1994 in France. We performed an ultrastructural comparative study between both virus-like particles found respectively in Pacific oysters and European flat oysters and compared them to viruses belonging to the Herpesviridae family. MATERIALS AND METHODS Specimens.Larval Ostrea edulis and Crassostrea gigas, 6 to 10 d old, 80 to 150 µm in size, were collected in May 1994 from 2 hatcheries located in Bourgneuf Bay (Vendée, France). Nursery-cultured spat of C. gigas and O. edulis, 3 to 5 mo old, were also collected from Bourgneuf Bay in June and July 1994.Light microscopy. A total of 197 European flat oyster spat and 196 Pacific oyster spat were collected for light microscopical examination. Moribund cultured spat of both species, 3 to 5 mo old, were removed from the shell and, sagittally sectioned; then half of each specimen was fixed in Davidson's fixative for light microscopic examination and the other half in Carson's fixative for transmission electron microscopic analysis. After 48 h fixation in Davidson's fluid, samples were dehydrated using an ...

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. KEY WORDS: Bonamia spp. · Real-time TaqMan PCR assay · Oyster Resale or republication not permitted without written consent of the publisherDis Aquat Org 71: [75][76][77][78][79][80] 2006 known to harbour Bonamia sp., an endemic isolate that has caused mortalities in the states of Victoria, Tasmania and Western Australia (Hine 1996, CochennecLaureau et al. 2003, Diggles 2003. Other isolates of Bonamia have been reported from O. chilensis in Chile (Campalans et al. 2000), Crassostrea ariakensis in North Carolina (Burreson et al. 2004) and O. puelchana in Argentina (Kroeck & Montes 2005). Taxonomic relationships between these isolates and described species within the genus need to be established.Although recent developments of PCR-based assays have partially addressed the limitations of histology (Carnegie & Cochennec-Laureau 2004), real-time PCR (polymerase chain reaction) has the potential to provide rapid and quantitative results. In order to establish an effective diagnostic capability that will help prevent the introduction of exotic Bonamia spp. and to avoid the spread of enzootic isolates, the development of a molecular-based diagnostic assay that allows rapid, reliable and sensitive detection of Bonamia spp. is required. This report describes the development of a real-time PCR assay capable of detecting Bonamia isolates with greater sensitivity than currently available methods. MATERIALS AND METHODSIsolates of Bonamia spp. Wild flat oysters Ostrea angasi, used as a source of Bonamia sp.-infected tissue, were collected and fixed in 95% ethanol, during the course of a survey on the health and genetics of stocks in 5 estuaries on the south coast of New South Wales, Australia (Heasman et al. 2004). European flat oyster O. edulis tissues, infected with isolates of B. ostreae and fixed in 95% ethanol, were obtained from France (6 samples) and the Netherlands (2 samples). Four bluff oyster O. chilensis tissues, infected with B. exitiosa and fixed in 95% ethanol, were obtained from New Zealand. In addition, DNA prepared from Bonamia-infected oysters O. edulis (Canada and USA) and O. chilensis (Chile) was included in the study. Furthermore,...

Two cases of haplosporidian infection occurred during 1993 in Pacific oystersCrassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oysterinfecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni. KEY WORDS: Pacific oyster · Crassostrea gigas · Haplosporidiosis · Haplosporidium nelsoniResale or republication not permitted without written consent of the publisher

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°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°C compared to 4°C and 15°C. Moreover, high salinities (≥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.

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.

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy. Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.

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 µm 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. KEY WORDS: Bonamia spp. · Ostrea edulis · Molecular analysis · Electron microscopy Resale or republication not permitted without written consent of the publisherDis Aquat Org 89: [79][80][81][82][83][84][85] 2010 Bonamia exitiosa infects Ostrea chilensis in New Zealand (Hine et al. 2001) and O. angasi in Australia (Corbeil et al. 2006) and has been recently reported in O. edulis in Spain (Abollo et al. 2008). B. roughleyi, previously called Mikrocytos roughleyi, infects Saccostrea glomerata in southeast Australia (Farley et al. 1988). B. perspora is a newly described protozoan species found in Ostreola equestris (North Carolina, USA) and represents the first Bonamia species producing a typical haplosporidian spore (Carnegie et al. 2006). Other B. exitiosa-like organisms have been described in O. chilensis from Chile (Kern 1993, Campalans et al. 2000, O. puelchana from Argentina (Kroeck & Montes 2005), and Crassostrea ariakensis from North Carolina ); these organisms have been subsequently declared B. exitiosa (Lopez-Flores et al. 2007).Previous studies have reported by microscopic and ultrastrustural means the occurrence in Italy of small parasites similar to Bonamia sp. in flat oysters collected from the southern (Tiscar et al. 1991) and northern Adriatic Sea (Tiscar et al. 2002). A small number of molluscs appeared infected by the parasites (1/161 from the southern and 8/600 from the northern Adriatic Sea, respectively). Nevertheless all the infected oysters showed high infection level of the haemocytes with the presence of numerous protozoans per cell (Tiscar et al. 1991(Tiscar et al. , 2002.Taking into account these previous reports, the aim of the present study was to investigate by cytology and histology the occurrence of microcell parasites in flat oysters Ostrea edulis newly collected from natural beds in the southern Adriatic Sea and to characterize the recovered parasites by molecular and ultrastructural analyses as well as to confirm and characterize the presence of Bonamia sp. in one positive sample collected during 1990 from the Manfredonia Gulf (Tiscar et al. 1991). MAT...