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

International audience; Disseminated neoplasia (DN) is a disease that affects bivalves worldwide and can lead to mass mortalities. In the present study, a pathological survey conducted from December 2011 to August 2012 in Crassostrea gasar, an oyster of commercial interest in northeast Brazil, revealed the occurrence of DN in oysters reared in the Mamanguape estuary, Paraiba State, Brazil. The present work describes the pathological and functional aspects of the disease in C. gasar by light microscopy (haemolymph cell monolayer and histological section) and flow cytometry analyses. The prevalence of the disease was low (7.1% of 182 oysters examined). Enlarged (neoplastic) cells showed reniform, ovoid or circular-shaped nuclei, with prominent nucleoli and predominantly short filipodia. They were found in the haemolymph and infiltrated the connective tissues of different organs, including the digestive system, gills and gonads, as well as in the sinuses and vessels. Three levels of progression of DN in tissues were observed, light (61.5%), moderate (15.4%) and advanced (23.1%). The viability of neoplastic cells circulating in the haemolymph (97.4%) was similar to that in the haemocytes (95.7%). The neoplastic cells showed low phagocytic ability (3.9%) compared with that of haemocytes (42.4%). Conversely, reactive oxygen species production (679 A.U.) and the total haemocyte count (3.9 x 10(6) cells mL(-1)) were higher in the affected oysters than in unaffected oysters (268 A.U. and 1.5 x 10(6) cells mL(-1), respectively). The low prevalence and primarily mild intensity found in the sampled oysters does not preclude an impact at the population level. A timely survey of DN is thus recommended in order to assess the severity and impact of this disease in wild and cultured populations of C. gasar oysters.

The spread, emergence, and adaptation of pathogens causing marine disease has been problematic to fisheries and aquaculture industries for the last several decades creating the need for strategic management and biosecurity practices. The Pacific oyster (Crassostrea gigas), a highly productive species globally, has been a target of disease and mortality caused by a viral pathogen, the Ostreid herpesvirus 1 (OsHV-1) and its microvariants (OsHV-1 µvars). During routine surveillance to establish health history at a shellfish aquaculture nursery system in San Diego, California, the presence of OsHV-1 in Pacific oyster juveniles was detected. Quantification of OsHV-1 in tissues of oysters revealed OsHV-1 viral loads > 106 copies/mg. We characterized and identified the OsHV-1 variant by sequencing of ORFs 4 (C2/C6) and 43 (IA1/IA2), which demonstrated that this variant is a novel OsHV-1 microvariant: OsHV-1 µvar SD. A pilot transmission study indicates that OsHV-1 µvar SD is infectious with high viral loads ~ 7.57 × 106 copies/mg detected in dead individuals. The detection of OsHV-1 µvar SD in a large port mirrors previous studies conducted in Australia where aquaculture farms and feral populations near port locations may be at a higher risk of OsHV-1 emergence. Further research is needed to understand the impacts of OsHV-1 µvar SD, such as transmission studies focusing on potential vectors and characterization of virulence as compared to other OsHV-1 µvars. To increase biosecurity of the global aquaculture industry, active and passive surveillance may be necessary to reduce spread of pathogens and make appropriate management decisions.

Ostreid herpesvirus-1 microvariants (OsHV-1) cause severe mortalities in farmed Crassostrea gigas in Europe, New Zealand and Australia. Outbreaks are seasonal, recurring in the warmer months of the year in endemic estuaries. The reference genotype and microvariant genotypes of OsHV-1 have been previously detected in the tissues of apparently healthy adult oysters naturally exposed to OsHV-1 in the field. However, the role of such oysters as reservoirs of infection for subsequent mortality outbreaks remains unclear. The aims of this study were: (1) to identify the optimal sample type to use for the detection of OsHV-1 DNA in apparently healthy C. gigas; and (2) to assess whether live C. gigas maintained on-farm after an OsHV-1 related mortality event remain infected and could act as a reservoir host for subsequent outbreaks. OsHV-1 DNA was detected in the hemolymph, gill, mantle, adductor muscle, gonad and digestive gland of apparently healthy adult oysters. The likelihood of detecting OsHV-1 DNA in hemolymph was equivalent to that in gill and mantle, but the odds of detecting OsHV-1 DNA in hemolymph and gill were more than 8 times that of adductor muscle. Gill had the highest viral loads. Compared to testing whole gill homogenates, testing snippets of the gill improved the detection of OsHV-1 DNA by about four fold. The prevalence of OsHV-1 in gill and mantle was highest after the first season of OsHV-1 exposure; it then declined to low or negligible levels in the same cohorts in subsequent seasons, despite repeated seasonal exposure in monitoring lasting up to 4years. The hemolymph of individually identified oysters was repeatedly sampled over 15months, and OsHV-1 prevalence declined over that time frame in the youngest cohort, which had been exposed to OsHV-1 for the first time at the start of that season. In contrast, the prevalence in two cohorts of older oysters, which had been exposed to OsHV-1 in prior seasons, was consistently low (

This study investigated Perkinsus spp. infecting Crassostrea rhizophorae from the Jaguaribe River estuary, Ceara, Brazil. Fragments of gills and rectum of the oysters (n = 150) were incubated in Ray’s fluid thioglycollate medium (RFTM). Genus Perkinsus-specific PerkITS85/750 PCR assays were performed and their amplicons were sequenced by the Sanger method. The RFTM assays confirmed Perkinsus spp. The sequencing of the amplified fragments from the rDNA internal transcribed spacers (ITS) of Perkinsus spp. confirmed Perkinsus chesapeaki. Neighbor-Joining analyzes place P. chesapeaki identified in this study in a well-supported clade with other isolates of the same species. This is the first record of P. chesapeaki infecting C. rhizophorae in South America.

The protozoan parasite Perkinsus marinus is the etiological agent of "dermo disease". This pathogen is considered by the World Organization for Animal Health (OIE) as reportable due to the high mortalities that it produces in the eastern oyster Crassostrea virginica in the US. In 2006, this parasite was detected in the pleasure oyster Crassostrea corteziensis in Nayarit on the Pacific coast of Mexico, indicating a new host and an extension of its known distribution. Epizootiological data of P. marinus in the pleasure oyster are unknown. With the objective of determining the prevalence and intensity in relation with temperature and salinity throughout time, as well as for studying interactions of host size and sex with the parasite, a monthly sampling was carried out in two aquaculture sites of Nayarit from 2007 to 2014. A total of 7700 oysters were analyzed. In both localities, prevalence was low in winter (6%) when temperature and salinity fluctuated around 24°C and 33, respectively; and the highest prevalence values occurred during summer (37%) when temperature and salinity were around 30°C and 20, respectively. Infection intensity increased in summer, but severe cases remained on average10%. Larger oysters showed the highest prevalence and intensity, and higher prevalence were generally observed in females. No unusual mortalities directly related with P. marinus were observed.

During routine histological examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, USA, a high prevalence and intensity of ciliate xenomas has been noted since sampling began in 1997. Xenomas are hypertrophic lesions on the gills of bivalve molluscs caused by intracellular ciliates, likely Sphenophrya sp. Although not known to cause mortality in oysters, xenomas have not previously been reported at this high abundance. The objectives of this study were to characterize the xenomas, describe the ciliates, and gather baseline epizootiological data with correlations to environmental and biological parameters. Upon gross examination, xenomas appeared as white nodules, up to 3mm in diameter, located in the gill tissue and occasionally fusing into large masses along the gill filaments. Light microscopy of histological sections revealed xenomas located in the gill water tubes, which they often completely blocked. Higher magnification revealed dual nuclei, eight kineties, and conjugation of the ciliates. Transmission electron microscopy revealed dual nuclei that varied in density, a maximum of twenty cilia in each kinety radiating from the oral apparatus to the posterior, and a 9+2 axoneme structure within the cilia. These traits place the ciliates into the Order Rhynchodida, but insufficient molecular data exist to confirm classification of this ciliate to the Genus Sphenophrya. Since 1997, xenoma prevalence has fluctuated with peaks in 2000, 2004, and 2011. Infected oysters generally contained30 xenomas, but 2.1% contained100, sharply contrasting the rare prevalence and low intensity reported elsewhere. Prevalence increased with oyster size, leveling off near 50% in oysters60mm. Infection intensity peaked in 70-90mm oysters and declined in larger oysters. Individual oyster condition was not associated with xenoma intensity, but sites with oysters in higher condition generally had a greater prevalence and intensity of xenoma infections. Seasonal data indicated an infection cycle increasing from summer to fall, peaking at 55-65% in November and dropping to10% by spring. The oyster population in Great Bay, NH warrants further examination to understand the mechanisms and conditions controlling xenoma formation.

High mortality rates are reported in spat and larvae of Pacific oyster Crassostrea gigas and associated with ostreid herpesvirus 1 (OsHV-1) detection in France. Although the viral infection has been experimentally reproduced in oyster larvae and spat, little knowledge is currently available concerning the viral entry and its distribution in organs and tissues. This study compares OsHV-1 DNA and RNA detection and localization in experimentally infected oysters using two virus doses: a low dose that did not induce any mortality and a high dose inducing high mortality. Real time PCR demonstrated significant differences in terms of viral DNA amounts between the two virus doses. RNA transcripts were detected in oysters receiving the highest dose of viral suspension whereas no transcript was observed in oysters injected with the low dose. This study also allowed observing kinetics of viral DNA and RNA detection in different tissues of oyster spat. Finally, viral detection was significantly different in function of tissues (p < 0.005), time (p < 0.005) with an interaction between tissues and time (p < 0.005) for each probe.

Perkinsus marinus, a World Organisation for Animal Health (OIE) notifiable parasite, infects several species of oyster, including Crassostrea virginica and Crassostrea corteziensis. There is little information on possible treatments for this parasite, but the biocidal properties of silver nanoparticles (AgNP) suggest their potential use. The lethal effects of the Argovit™ formulation of AgNP was evaluated for the first time against hypnospores of P. marinus, a particularly resistant stage of the parasite that persists in the environment until favorable conditions occur for zoosporulation to be induced. Hypnospores were exposed to 1, 10 and 100 µg/mL of silver compounded in Argovit™ (corresponding to 0.009, 0.093 and 0.927 mM of Ag), to 157.47 µg/mL (0.927 mM) of silver nitrate (AgNO3) used as a positive control, and to polyvinylpyrrolidone (PVP, 1570 µg/mL) used as a vehicle control. Hypnospores in culture medium without treatment served as a negative control. Dose-dependence after 24 h of exposure to AgNP was observed. A concentration of 0.093 mM AgNP resulted in 50% mortality of P. marinus. Treatment with 0.927 mM of silver, as AgNP or AgNO3, was highly lethal, with greater than 90% mortality. Silver nanoparticles were implicated in the deformation of hypnospores. Transmission electron microscopy (TEM) revealed AgNP within the hypnospore wall and involved in the degradation of lipid droplets in the cytoplasm. AgNP were effective in a saline medium, suggesting the utility of detailed studies of the physicochemical interactions of AgNP under these conditions. These results suggest investigations of possible effect of Argovit™ formulation of AgNP against stages of the parasite like trophozoites and tomonts that develop in tissues or hemolymph of infected oysters as well as studies on its effects in the host and environment.

International audience; 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.

Oyster species suffer from numerous disease outbreaks, often causing high mortality. Because the environment cannot be controlled, genetic improvement for disease resistance to pathogens is an attractive option to reduce their impact on oyster production. We review the literature on selective breeding programs for disease resistance in oyster species, and the impact of triploidy on such resistance. Significant response to selection to improve disease resistance was observed in all studies after two to four generations of selection for Haplosporidium nelsoni and Roseovarius crassostrea in Crassostrea virginica, OsHV-1 in Crassostrea gigas, and Martelia sydneyi in Saccostrea glomerata. Clearly, resistance in these cases was heritable, but most of the studies failed to provide estimates for heritability or genetic correlations with other traits, e.g., between resistance to one disease and another. Generally, it seems breeding for higher resistance to one disease does not confer higher resistance or susceptibility to another disease. For disease resistance in triploid oysters, several studies showed that triploidy confers neither advantage nor disadvantage in survival, e.g., OsHV-1 resistance in C. gigas. Other studies showed higher disease resistance of triploids over diploid as observed in C. virginica and S. glomerata. One indirect mechanism for triploids to avoid disease was to grow faster, thus limiting the span of time when oysters might be exposed to disease.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

Bivalves are filter feeders that obtain food from seawater that may contain infectious agents, such as the protozoan parasites Perkinsus marinus and P. olseni that are associated with massive mortalities responsible for losses in the aquaculture industry. Despite all physical and chemical barriers, microorganisms cross epithelia and infect host tissues to cause pathologies. Epigenetics mechanisms play important roles in a variety of human processes, from embryonic development to cell differentiation and growth. It is currently emerging as crucial mechanism involved in modulation of host-parasite interactions and pathogenesis, promoting discovery of targets for drug treatment. In bivalves, little is known about epigenetic mechanism in host parasite interactions. The objective of the present study was to evaluate the effect of Perkinsus sp. infections on DNA methylation levels in tissues of Crassostrea gasar oysters. Samples were collected in 2015 and 2016 in the Mamanguape River estuary (PB). Oyster gills were removed and used for Perkinsus sp. diagnosis. Gills (G) and gastrointestinal tract (GT), as well as cultured P. marinus trophozoites were preserved in 95% ethanol for DNA extractions. DNA methylation levels were estimated from G and GT tissues of uninfected (n = 60) and infected oysters (n = 60), and from P. marinus trophozoites, by ELISA assays. Results showed that the mean prevalence of Perkinsus sp. infections was high (87.3%) in 2015 and moderate (59.6%) in 2016. DNA methylation levels of G and GT tissues were significantly lower in infected oyster than in uninfected oysters, suggesting that infections are associated with hypomethylation. Methylation level was significantly higher in G than in GT tissues, indicating a likely tissue-specific mechanism. P. marinus trophozoites showed 33% methylation. This was the first study that confirms alterations of DNA methylation in two tissues of C. gasar oysters in association with Perkinsus sp. infections.

Extracellular products (ECPs) of the French V. tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1, 10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Even if taken together, our results let us wonder if this (these) zinc metalloprotease(s) could be involved in the impairment of hemocyte immunological functions, the direct implication of this (these) protein(s) in ECPs toxicity still has to be demonstrated.

Massive mortality outbreaks have been reported in France since 2008 among Pacific oysters, Crassostrea gigas, with the detection of a particular OsHV-1 variant called μVar. Virus infection can be induced in healthy spat in experimental conditions allowing to better understand the disease process, including viral gene expression. Although gene expression of other herpesviruses has been widely studied, we provide the first study following viral gene expression of OsHV-1 over time. In this context, an in vivo transcriptomic study targeting 39 OsHV-1 genes was carried out during an experimental infection of Pacific oyster spat. For the first time, several OsHV-1 mRNAs were detected by real-time PCR at 0 h, 2 h, 4 h, 18 h, 26 h and 42 h post injection. Several transcripts were detected at 2 h post infection and at 18 h post infection for all selected ORFs. Quantification of virus gene expression at different times of infection was also carried out using an oyster housekeeping gene, Elongation factor. Developing an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral gene appears a new tool in terms of diagnosis and can be used to complement viral DNA detection in order to monitor viral replication.

Vibrio coralliilyticus infects a variety of shellfish larvae, including Pacific oyster (Crassostrea gigas) larvae worldwide, and remains a major constraint in marine bivalve aquaculture practice, especially in artificial seed production facilities. In this study, we isolated and characterized the bacteriophage (phage) that specifically infects V. coralliilyticus. The phage was designated pVco-14 and classified as Siphoviridae. We also investigated the potential efficacy of the isolated phage against V. coralliilyticus infection. We conducted a survey to replace the overuse of antibiotics, which generate multi-antibiotic-resistant strains and causes environmental pollution. The latent period of pVco-14 was estimated to be approximately 30 min, whereas the burst size was 13.3 PFU/cell. The phage was found to infect four strains of tested V. coralliilyticus. pVco-14 was stable at wide temperature (4–37 °C) and pH (5.0–9.0) ranges. Eighty-one percent of oyster larvae died in an immersion challenge at a dose 1.32 × 105 CFU/ml of virulent V. coralliilyticus (strain 58) within 24 h. When oyster larvae were pre-treated with the phage before the bacterial challenge (bacterial conc.: 1.32 × 104 and 1.32 × 105 CFU/ml), mortality of the phage-treated oyster larvae was lower than that of the untreated control. These results suggest that pVco-14 has potential to be used as a prophylactic agent for preventing V. coralliilyticus infection in marine bivalve hatcheries and can reduce the overuse of antibiotics.

Rickettsia infection in adult Japanese oysters (Crassostrea gigas) was observed in 2015 at San Ignacio Lagoon, Baja California Sur, Mexico and characterized using molecular tools. In the present study, the degree of infection by Eosinophilic Rickettsia-Like Organism (E-RLO), characterized by intracellular inclusions in gill, mantle, labial palps, digestive tract and gonadal ducts, was evaluated using histological methods and was associated with visible injuries on body surface, such as blisters, shell damage and necrosis. Most of the oysters (92.2%) had some type of observable symptoms; 90.2% of all oysters had blisters in the mantle, and all oysters presented with E-RLO in the epithelia of at least one of the analyzed tissues (gills, labial palps, mantle, gonadal ducts and digestive tract). The highest intensity of infection (percent of E-RLO coverage) was observed in the labial palps (18.6%) and the lowest in digestive tract (2.6% of the examined tissue). Oysters with external injuries had higher infection intensities than the oysters without external injuries. Considering the clinical signs (observable symptoms and histological findings), we propose three infective stages of E-RLO disease development, a scale that could be used for early detection.

In order to determine if ostreid herpesvirus 1 (OsHV-1) can be vertically transmitted, 9 full-sib families of the Portuguese oyster Crassostrea angulata were produced using a factorial mating design with 3 males and 3 females. The parents were survivors from an OsHV-1 mortality outbreak. OsHV-1 DNA was not detected by conventional PCR in the mantle of parents, gametes or 3 day-old larvae. However, viral DNA was detected by real-time PCR in all gametes and larvae samples. These results show that C. angulata that have survived an OsHV-1 mortality outbreak can carry the virus and vertically transmit it to their offspring.

French production of the Pacific cupped oyster, Crassostrea gigas, is currently threatened by two pathogens, OsHV-1 and V. aestuarianus. While oysters selected for their higher resistance to OsHV-1 are now available for the industry, the impact of V. aestuarianus on such oysters is unknown, especially for triploids. In addition, experimental infection has used the virus or the bacteria alone, but there have been no investigations of dual exposure to these pathogens. This study is the first report of single or dual exposure in spat (Spat1 and Spat2), juvenile and adult naïve oysters. For each of the two stocks evaluated, unselected oysters and oysters selected for their higher resistance to OsHV-1 infection were tested, as well as their triploid siblings of the selected oysters produced using cytochalasin B. We confirmed that resistance to OsHV-1 infection and susceptibility to V. aestuarianus increased with age and size, although selected oysters were not significantly impacted by OsHV-1 whatever their ploidy, size or age. We found different mortality patterns depending on the pathogen tested. The mortality pattern was similar for oysters exposed to OsHV-1 or to both pathogens in the Spat1 trial (4 months old and 1.9 g). The mortality pattern was similar for oysters exposed to V. aestuarianus or to both pathogens in the Adult trial (25 months old and 63.1 g). Surprisingly, mortality was much higher (ranging from 75.9% to 100%), in particular for the selected oysters, for the Spat2 (8 months old/3.9 g) and Juvenile trials (16 months old/18.4 g) given a dual exposure, regardless of the level of selection for OsHV-1 and the ploidy state. Our findings highlight an important threat for oyster farmers: oysters exposed to both pathogens could experience dramatic mortality rates, even in oysters selected for their higher resistance to OsHV-1. Finally, our study demonstrated for the first time that triploid oysters were more susceptible to experimental challenges with V. aestuarianus at the spat stage than their diploid siblings. However, the difference in mortality between the triploids and diploids remained limited and ranged from 22.9% to 6.6% for spat and adults, respectively with a relatively regularly decrease in the difference with increased age.

In Galicia, there is an increasingly interest among representatives of the oyster industry in the development of Pacific oyster Crassostrea gigas culture. Nevertheless severe mortalities and emerging health problems in this species have been recently reported in European farming areas. A histological survey was performed from 2004 to 2009 to assess health status in both cultured and wild Galician oysters. Different symbiotic organisms and conditions were detected, including viral gametocytic hypertrophy (VGH) which is reported here for first time in Spanish coast. VGH, prokaryote-like colonies and ciliates were observed in oyster tissues without causing host damage. A haplosporidian infection, copepods inducing lesions and a cellular proliferative disorder were detected in some samples causing moderate host damage; their low prevalence suggests these parasites are not a threat for C. gigas in Galicia. None of the parasites detected is OIE (Office International des Epizooties: the World Organization for Animal Health) notifiable. Although the current study did not identify any pathogens or diseases of concern, it provides important prevalence baseline data for future health and epidemiological assessments needed to better understanding the existing and emerging health problems in this species.

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a "lumpy" appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 degrees C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 degrees C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 degrees C: 0% viability), 60 days (3.5 ppt, 10 degrees C: 0% viability) and 90 days (7 ppt, 2 degrees C: 0.6+/-0.7%; 7 ppt, 10 degrees C: 0.2+/-0.2%).

The protistan parasite Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), induces mortality and reduces marketability in the Pacific oyster, Crassostrea gigas, in British Columbia, Canada. This parasite is a pathogen of international concern because it infects a range of oyster species, and because its life cycle and mode of transmission are unknown. A digoxigenin-labelled DNA probe in situ hybridisation technique (DIG–ISH) was developed, and its detection sensitivity was compared to standard histological sections stained with haematoxylin and eosin stain (H&E-histo). In H&E-histo preparations, the detection of M. mackini was certain only when the parasite occurred within the vesicular connective tissue of adult oysters. However, the DIG–ISH technique clearly demonstrated the presence of infection in all other host tissues as well as in juvenile oysters with poorly developed vesicular connective tissue. The probe hybridised strongly to M. mackini, did not hybridise to oyster tissues or with the other shellfish parasites tested, and was more sensitive for detecting infections when compared to H&E-histo.

Viral infection caused by Ostreid herpesvirus 1 (OsHV-1) is one of the proximate causes of mass mortalities of cultivated bivalves around the world. The emergence and spread of different variants of OsHV-1 accompanied by different epidemiological characteristics have been reported frequently in different countries around the world. In this paper, we present a study of the detection of OsHV-1 DNA and their variations from 1599 samples over 18 species collected in 27 aquaculture sites and two food markets during 2001–2013 in China. All of the samples were examined by a nested PCR assay targeting the C2/C6 fragment of OsHV-1 followed by sequencing. Our results showed 338 individuals (21.1%) of seven species sampled from 14 (14/27 = 51.9%) sites and the two food markets were positive for viral DNA. Sequencing of 289 PCR products revealed 24 virus types. No shared virus type was found among different countries with 47 types (23 in Japan, 16 in France, 2 in South Korea and 1 in each country of Australia, USA, Ireland, New Zealand, Mexico and China) identified in previous studies. As previously reported, two main phylogenetic groups were identified by phylogenetic analysis based on the 71 virus types; within which 6 separate clades were identified. Our results also demonstrated that two clades were associated with abnormal mortalities of the scallop, Chlamys farrier and the calm, Scapharca broughtonii in China. These findings indicated that cultivated bivalves may face potential threats from OsHV-1 types found in our study.

© 2015 Elsevier Inc. Effects of experimental exposure to Alexandrium fundyense, a Paralytic Shellfish Toxin (PST) producer known to affect bivalve physiological condition, upon eastern oysters, Crassostrea virginica with a variable natural infestation of the digenetic trematode Bucephalus sp. were determined.After a three-week exposure to cultured A. fundyense or to a control algal treatment with a non-toxic dinoflagellate, adult oysters were assessed for a suite of variables: histopathological condition, hematological variables (total and differential hemocyte counts, morphology), hemocyte functions (Reactive Oxygen Species (ROS) production and mitochondrial membrane potential), and expression in gills of genes involved in immune responses and cellular protection (MnSOD, CAT, GPX, MT-IV, galectin CvGal) or suspected to be (Dominin, Segon).By comparing individual oysters infested heavily with Bucephalus sp. and uninfested individuals, we found altered gonad and digestive gland tissue and an inflammatory response (increased hemocyte concentration in circulating hemolymph and hemocyte infiltrations in tissues) associated with trematode infestation. Exposure to A. fundyense led to a higher weighted prevalence of infection by the protozoan parasite Perkinsus marinus, responsible for Dermo disease. Additionally, exposure to A. fundyense in trematode-infested oysters was associated with the highest prevalence of P. marinus infection. These observations suggest that the development of P. marinus infection was advanced by A. fundyense exposure, and that, in trematode-infested oysters, P. marinus risk of infection was higher when exposed to A. fundyense. These effects were associated with suppression of the inflammatory response to trematode infestation by A. fundyense exposure. Additionally, the combination of trematode infestation and A. fundyense exposure caused degeneration of adductor muscle fibers, suggesting alteration of valve movements and catch state, which could increase susceptibility to predation. Altogether, these results suggest that exposure of trematode-infested oysters to A. fundyense can lead to overall physiological weakness that decrease oyster defense mechanisms.

This study described seasonal differences in the histopathological and hemolymph chemistry changes in different family lines of Pacific oysters, Crassostrea gigas, in response to the stress of an abrupt change to low salinity, and mechanical grading. The most significant changes in pallial cavity salinity, hemolymph chemistry and histopathological findings occurred in summer at low salinity. In summer (water temperature 18°C) at low salinity, 9 (25.7% of full salinity), the mean pallial cavity salinity in oysters at day 3 was 19.8±1.6 (SE) and day 10 was 22.8±1.6 (SE) lower than oysters at salinity 35. Associated with this fall in pallial cavity salinity, mean hemolymph sodium for oysters at salinity 9 on day 3 and 10 were 297.2mmol/L±20(SE) and 350.4mmol/L±21.3(SE) lower than oysters at salinity 35. Similarly mean hemolymph potassium in oysters held at salinity 9 at day 3 and 10 were 5.6mmol/L±0.6(SE) and 7.9mmol/L±0.6 (SE) lower than oysters at salinity 35. These oysters at low salinity had expanded intercellular spaces and significant intracytoplasmic vacuolation distending the cytoplasm of epithelial cells in the alimentary tract and kidney and hemocyte infiltrate (diapedesis) within the alimentary tract wall. In contrast, in winter (water temperature 8°C) oyster mean pallial cavity salinity only fell at day 10 and this was by 6.0±0.6 (SE) compared to that of oysters at salinity 35. There were limited histopathological changes (expanded intercellular spaces and moderate intracytoplasmic vacuolation of renal epithelial cells) in these oysters at day 10 in low salinity. Mechanical grading and family line did not influence the oyster response to sudden low salinity. These findings provide additional information for interpretation of non-lethal, histopathological changes associated with temperature and salinity variation.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60–90% in oysters

Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.

In order to develop an alternative method to antibiotics for preventing bacillary necrosis in bivalve mollusc larvae, we examined the effects of ovoglobulins (proteins derived from the whites of hens' eggs) on the survival of larvae of the Pacific oyster Crassostrea gigas. The pathogenic Vibrio tubiashii (ATCC 19106) was used to infect larvae of the Pacific oyster. V. tubiashii showed strong pathogenicity to oyster larvae, causing 100% mortality after experimental exposure for 24 h at a concentration of 10(5) cfu (colony-forming units)/ml. In contrast, the addition of ovoglobulins at a concentration of 10 microg/ml to larval oysters, challenged with V. tubiashii at 10(5) cfu/ml, led to a marked increase in larval survival of 96.5% at 24 h after infection. The V. tubiashii culture supernatant was also shown to be pathogenic to larval oysters; however, its pathogenicity was completely inhibited by the addition of 10 microg/ml of ovoglobulins. Larval oysters infected by V. tubiashii showed typical symptoms of bacillary necrosis including anomalous swimming and detachment of cilia and/or vela. In contrast, live larvae were actively motile, and their cilia and vela were not necrotized in the ovoglobulins-added group. The addition of ovoglobulins clearly suppressed the growth of V. tubiashii in gelatin-sea water broth, but the number of viable V. tubiashii 24 h after incubation did not decrease to the initial dose level. Findings obtained in this study indicate that ovoglobulins almost completely protect larval oysters from V. tubiashii infection by nonbactericidally inhibiting the growth of V. tubiashii without affecting survival of the oysters.

Hemocytes play an important role in internal defence in molluscs. The morphology of hemolymph cells was studied for the first time in Ruditapes decussatus. Two main types of hemocytes (hyalinocytes and granulocytes) exist in R. decussatus. Three types of granulocytes were identified by light microscopy, in accordance with the presence of basophilic or acidophilic granules or a mixture of both in the cytoplasm. The existence of hyalinocytes and granulocytes was confirmed by electron microscopy. Some monoclonal antibodies (MABs) raised against hemocytes of Crassostrea gigas showed cross-reactivity with the total population of hemocytes of R. decussatus; however, none of the MABs raised against hemocytes of Mytilus edulis showed cross-reactivity. The MABs assayed did not allow us to distinguish hemocyte subpopulations.

Brazilian production of bivalve molluscs is small but expanding, especially in the northeastern region where the native oysters Crassostrea rhizophorae and C. gasar are abundant, and tropical weather promotes their rapid growth. Studies on bivalve pathology are scarce in Brazil, with only a few employing techniques for detecting protozoan pathogens listed by the World Organisation for Animal Health (OIE). In 2008, a Perkinsus sp. was reported for the first time in Brazil, infecting C. rhizophorae oysters from a wild population in Ceara state, NE Brazil. Recently P. marinus was detected in the same oyster species in nearby Paraiba state. These findings highlighted the need to expand knowledge on the presence and impacts of Perkinsus spp. on Brazilian oyster populations. The current investigation evaluated Perkinsus sp. infections among wild and cultured C. gasar mangrove oysters from the estuary of the Rio Sao Francisco, Sergipe state, NE Brazil. Our results show that Perkinsus sp. infections occurred commonly in oysters of both groups, at prevalences that were frequently higher among cultured oysters. Prevalences varied seasonally, with maximum values during summer (January) of 57% and 80% for wild and cultured oysters respectively, and minimum values during winter (July). Results of DNA sequencing, in situ hybridization assays, and phylogenetic analyses showed dual- and single-pathogen infections by P. marinus and/or P. olseni in the tested oysters.

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.

Denman Island disease, characterized by clinical signs of focal green lesions on the body surface or within the mantle, palps, and adductor muscle of Pacific oysters (Crassostrea gigas), is caused by a protozoan parasite of unknown taxonomic affiliations, Mikrocytos mackini. Detection of M. mackini was more sensitive and rapid by use of tissue imprints than histological sections. Of several isolation procedures investigated, centrifugation of homogenized infected tissues through a 15% sucrose solution enabled the isolation of the highest number of M. mackini with the lowest amount of oyster debris. Experimental transmission showed that oysters exposed to M. mackini by inoculation with isolated parasites had shorter prepatent periods and higher prevalences and intensities of infection than those incubated in homogenates from infected oysters (bath exposure) or those naturally exposed in the field. Experimental transmission was also used to propagate M. mackini in vivo in the laboratory year round. For the development of the disease, exposed oysters required prolonged incubation at low temperatures (about 10 degrees C).

We have succeeded in establishing an in vitro continuous culture of the eastern oyster ( Crassostrea virginica ) parasite Perkinsus marinus (Gauthier and Vasta, 1993) and have now characterized a number of variables, such as temperature, salinity, pH, seeding density, and selected nutritional requirements that affect parasite proliferation. Optimum temperature, salinity, and pH ranges were 28-32°C, 25-30 ppt, and 6.6-6.8, respectively. Proliferation rates during the first 48 hr of culture were directly related to the size of the inoculum. The addition of Ham's F12 nutrient mixture (a serum replacement) to Dulbecco modified Eagle's (DME) base medium substantially enhanced proliferation, particularly at a DME:Ham's F12 ratio of 1:2. Five percent fetal bovine serum was required for optimal growth, but higher concentrations (10-20%) were inhibitory. The presence of oyster plasma enhanced growth only at low concentrations of fetal bovine serum (0-0.1%) and doubled addition of Ham's F12 (DME:Ham's 1:2). After approximately 30 passes over a 6-month period, the cultured parasite exhibited morphological and virulence features that are similar to those from the freshly collected specimens. The optimized medium apparently provides an environment comparable to the intracellular conditions of the host since the small (approximately 4 μm) trophozoite stage frequently observed to proliferate within the oyster hemocyte is maintained.

Hatchery-produced, unparasitized oysters (Crassostrea virginica) were transplanted to high and low salinity sites in the Chesapeake Bay in May 1992. These locations included Mobjack Bay, Virginia (MB, ∼20%), the Choptank River, Maryland (CR, ∼14%), and the Wye River, Maryland (WR, ∼8%); the salinities subsequently varied with rainfall in the region of each site. The protozoan parasite Perkinsus marinas rapidly infected the oysters at each site. Oysters were periodically sampled and individually assayed for intensity of P. marinus infection, total circulating hemocyte count (THC) and zymosan-iuduced chemiluminescence (CL) activity of the hemocytes. The CL assay measures the production of reactive oxygen intermediates (ROIs) thought to mediate antimicrobial activity of the hemocytes. The MB oysters were 100% infected and showed a high intensity of infection by October, 1992; during the following year, high levels of infection were reached rapidly and the population was completely decimated by mid-July, 1993. Disease intensity and progression were slower in oysters from the low salinity sites (CR and WR). The THC and zymosan-stimulated CL responses of hemocytes from moderately or heavily infected oysters were significantly higher than those of uninfected or lightly infected oysters. The increased number of hemocytes from oysters with advanced infections probably represents a general protective response to the presence of infectious agents in tbe hemolymph. Hemocytes withdrawn from oysters with heavy P. marinus infections have markedly increased CL activity after phagocytosis of zymosan particles. Statistical analysis of the data showed that this activated condition was strongly associated with the level of P. marinus infection of the host, but not influenced by the ambient salinity of the oysters' habitat. However, phagocytosis of P. marinus merozoites by hemocytes fails to trigger CL, regardless of the infection intensity of the oysters from which the hemocytes were withdrawn. These data suggest that ingestion of microorganisms may nonspecifically enhance hemocytic defense responsiveness, as measured by their increased production of ROIs upon subsequent phagocytic stimulation by zymosan. However, not all microbes can initiate responses from these activated hemocytes; this may partially explain the virtual inability of C. virginica hemocytes to control P. marinus infections by intracellular cidal mechanisms.