Rapid capture and detection of ostreid herpesvirus-1 from Pacific oyster Crassostrea gigas and seawater using magnetic beads.

Ostreid herpesvirus-1 (OsHV-1) has been involved in mass mortality episodes of Pacific oysters Crassostrea gigas throughout the world, causing important economic losses to the aquaculture industry. In the present study, magnetic beads (MBs) coated with an anionic polymer were used to capture viable OsHV-1 from two types of naturally infected matrix: oyster homogenate and seawater. Adsorption of the virus on the MBs and characterisation of the MB-virus conjugates was demonstrated by real-time quantitative PCR (qPCR). To study the infective capacity of the captured virus, MB-virus conjugates were injected in the adductor muscle of naïve spat oysters, using oyster homogenate and seawater without MBs as positive controls, and bare MBs and sterile water as negative controls. Mortalities were induced after injection with MB-virus conjugates and in positive controls, whereas no mortalities were recorded in negative controls. Subsequent OsHV-1 DNA and RNA analysis of the oysters by qPCR and reverse transcription qPCR (RT-qPCR), respectively, confirmed that the virus was the responsible for the mortality event and the ability of the MBs to capture viable viral particles. The capture of viable OsHV-1 using MBs is a rapid and easy isolation method and a promising tool, combined with qPCR, to be applied to OsHV-1 detection in aquaculture facilities.

Prevalences of pathogenic and non-pathogenic Vibrio parahaemolyticus in mollusks from the Spanish Mediterranean Coast.

Vibrio parahaemolyticus is a well-recognized pathogen of humans. To better understand the ecology of the human-pathogenic variants of this bacterium in the environment, a study on the prevalence in bivalves of pathogenic variants (tlh+ and tdh+ and/or trh+) versus a non-pathogenic one (only tlh+ as species marker for V. parahaemolyticus), was performed in two bays in Catalonia, Spain. Environmental factors that might affect dynamics of both variants of V. parahaemolyticus were taken into account. The results showed that the global prevalence of total V. parahaemolyticus found in both bays was 14.2% (207/1459). It was, however, significantly dependent on sampling point, campaign (year) and bivalve species. Pathogenic variants of V. parahaemolyticus (tdh+ and/or trh+) were detected in 3.8% of the samples (56/1459), meaning that the proportion of bivalves who contained tlh gene were contaminated by pathogenic V. parahaemolyticus strains is 27.1% (56/207). Moreover, the presence of pathogenic V. parahaemolyticus (trh+) was significantly correlated with water salinity, thus the probability of finding pathogenic V. parahaemolyticus decreased 1.45 times with every salinity unit (ppt) increased. Additionally, data showed that V. parahaemolyticus could establish close associations with Ruditapes spp. (P-value < 0.001), which could enhance the transmission of illness to human by pathogenic variants, when clams were eaten raw or slightly cooked. This study provides information on the abundance, ecology and characteristics of total and human-pathogenic V. parahaemolyticus variants associated with bivalves cultured in the Spanish Mediterranean Coast.

Update of information on perkinsosis in NW Mediterranean coast: Identification of Perkinsus spp. (Protista) in new locations and hosts.

This study addressed perkinsosis in commercially important mollusc species in the western Mediterranean area. Perkinsus olseni was found in Santa Gilla Lagoon (Sardinia) infecting Ruditapes decussatus, Cerastoderma glaucum and Venerupis aurea, in Balearic Islands infecting Venus verrucosa and in Delta de l'Ebre (NE Spain) parasitising Ruditapes philippinarum and R. decussatus. Perkinsus mediterraneus was detected infecting Ostrea edulis from the Gulf of Manfredonia (SE Italy) and Alacant (E Spain), V. verrucosa and Arca noae from Balearic Islands and Chlamys varia from Balearic Islands, Alacant and Delta de l'Ebre.

Uptake and retention of Vibrio parahaemolyticus in a cohabitating population of Ruditapes decussatus and Ruditapes philippinarum under experimental conditions.

The presence of Vibrio parahaemolyticus in bivalve mollusc is an important cause of foodborne illnesses, and their levels are influenced by environmental changes, such as temperature and salinity. Clams are common species in estuaries and are used in environmental monitoring programmes. Present study compared the uptake and retention of nonpathogenic V. parahaemolyticus by two species of clam (Ruditapes decussatus and R. philippinarum), cohabitating in a closed system. Results showed no significant differences were found between both species of clams. Bacterial levels are following a similar trend with values between 3.48 and 3.70 log CFU/g for R. decussatus and between 3.15 and 3.49 log CFU/g for R. philippinarum. So, in the absence of water renewal, high and stable levels of V. parahaemolyticus were observed in cultured clams after exposure. Changes in physical parameters should be taken into account to design surveillance programmes in bivalves, and sampling should focus on species that have faster filtration rates at that water temperature since they potentially represent the worst-case scenario.

Detection and identification of tdh- and trh-positive Vibrio parahaemolyticus strains from four species of cultured bivalve molluscs on the Spanish Mediterranean Coast.

Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.

First record of Perkinsus olseni, a protozoan parasite infecting the commercial clam Ruditapes decussatus in Spanish Mediterranean waters.

We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.

Mitigation of lethal effects of Karlodinium veneficum and K. armiger on Sparus aurata: changes in haematocrit and plasma osmolality.

Between January and April 2000, several experiments were performed during a Karlodinium spp. proliferation in Alfacs Bay (Ebro delta, NW Mediterranean) to determine the effects of these dinoflagellates on sea bream Sparus aurata cultivated in the area. Moribund fish showed an increase in plasma osmolality together with a decrease in the haematocrit percentage compared to control fish. The efficacy of copper sulphate, hydrogen peroxide, potassium permanganate and formalin against Karlodinium spp. was also tested. None of these treatments had mitigation effects when applied in the presence of fish; on the contrary, lethal effects appeared at lower Karlodinium spp. densities compared to fish control groups. When a lytic agent, such as copper sulphate, was used as a water pre-treatment, in the absence of fish, Karlodinium spp. toxicity was significantly reduced. Protocols for water pre-treatments were studied as a potential tool for combating Karlodinium spp. in fish farms.