Perkinsus chesapeaki observed in a new host, the European common edible cockle Cerastoderma edule, in the Spanish Mediterranean coast.

Histological observations showed the presence of a Perkinsus sp. parasite in Cerastoderma edule tissues for the first time in the Spanish Mediterranean coast. ITS molecular characterization by PCR-RFLP, in situ hybridization and sequencing, identified the parasite as Perkinsus chesapeaki, with a maximum identity of 99-100% with GenBank P. chesapeaki sequences from France and 97% with P. chesapeaki sequences of North American origin when BLAST analysis was carried out. Furthermore, phylogenetic studies placed the European cockle parasite in a well defined cluster together with the other European isolates. This is the first report of P. chesapeaki in the cockle C. edule.

Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast.

Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B. exitiosa was first detected, in 2007, on this continent in flat oysters cultured in Galicia (NW Spain). Since then, the parasite has also been detected in France, Italy and the United Kingdom. The bays of the Ebro Delta in the south of Catalonia represent the main bivalve culture area in the Mediterranean coast of Spain. Previous information from the area includes reports of several flat oyster pathogens, including the notifiable parasite Marteilia refringens. However, the status with regard to Bonamia parasites was uncertain. In the present study, a Bonamia parasite was observed in flat oysters cultured in the Alfacs Bay of the Ebro Delta by histology and real-time PCR. PCR-RFLP and sequencing suggested the presence of B. exitiosa. Finally, phylogenetic analyses of the studied Bonamia isolates corroborated B. exitiosa infection. M. refringens was also observed in the same oyster batch, and co-infection with both parasites was also detected. This is the first detection of B. exitiosa, in Catalonia and the Spanish Mediterranean coast. The impact of the parasite on the Mediterranean flat oyster activity needs to be urgently addressed.

Persistence of sucrose fermenting and nonfermenting vibrios in tissues of Manila clam species, Ruditapes philippinarum, depurated in seawater at two different temperatures.

Effective post-harvest treatment to eliminate toxigenic vibrios is an important measure to reduce risk associated with seafood consumption. In the present study, we examined whether natural populations of sucrose nonfermenting (SNFV) and sucrose fermenting vibrios (SFV) persisted in Ruditapes philippinarum, after depuration at two different temperatures. Two experiments (one in Spring and one in Summer) were performed. Clams were depurated in duplicate tanks in an open-circuit seawater system using filtration and ultraviolet treatment. Clams were sampled daily (9 days), individually processed and inoculated on thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates to estimate the density of SNFV and SFV. Results showed that depuration was less effective in Summer than in Spring, for SNFV and SFV. Despite an initial lower presence in the Summer experiment, clearance of SNFV was less efficient than in Spring, with 22.2% of clams presenting SNFV on day 8 and without significant changes on day 9. When compared, daily proportion of presence of SNFV and SFV in Spring, it was observed that SFV were more frequent than SNFV on days 8 and 9. In Summer these differences were significant on days 7-9. Present study demonstrated that SNFV (involved in food poisoning) are purged, at any temperature, faster than SFV.

Experimental uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in two species of clams: Ruditapes decussatus and Ruditapes philippinarum.

AIMS: To describe uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in Ruditapes decussatus and Ruditapes philippinarum. METHODS AND RESULTS: Ruditapes decussatus accumulated greater concentrations of pathogenic and nonpathogenic V. parahaemolyticus than R. philippinarum. These concentrations decreased earlier in R. decussatus. Nonpathogenic V. parahaemolyticus reached higher concentrations (approx. 1 log CFU g⁻¹ in tissues of R. decussatus and more than 1 log CFU g⁻¹ in R. philippinarum) than pathogenic V. parahaemolyticus at similar times. It also persisted longer in both species of clams (72 h in R. decussatus and 96 h in R. philippinarum), while pathogenic V. parahaemolyticus persisted 48 h in R. decussatus and 72 h in R. philippinarum. CONCLUSIONS: Ruditapes decussatus incorporated both isolates of V. parahaemolyticus faster than R. philippinarum and it eliminated both isolates earlier than R. philippinarum. Under same conditions, nonpathogenic V. parahaemolyticus might survive better than pathogenic V. parahaemolyticus within both species of clam. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathogenic V. parahaemolyticus is an important cause of foodborne illnesses. This study shows it may be possible to use nonpathogenic V. parahaemolyticus to perform experimentation to evaluate bacterial evolution in clams, developing an in vivo model useful for risk analysis.

Comparative experimental infection of the copepod Paracartia grani with Marteilia refringens and Marteilia maurini.

Paracartia grani (Copepoda) has been identified as a potential intermediate host in the life cycle of Marteilia refringens, a paramyxean parasite infecting flat oysters. However, no intermediate host has yet been identified for Marteilia maurini that infects mussels. A better understanding of the life cycle of these two Marteilia types would clarify their taxonomic relationship and hypothesized co-specificity. For this purpose, experimental infections of copepods, P. grani, were performed using naturally infected flat oysters and mussels. Infection patterns were different depending whether copepods were infected from oysters or mussels. M. maurini did not proliferate in copepods while M. refringens rapidly proliferated in infected copepods. Previously unrecognized developmental stages of M. refringens were found during this study.

Delta de l'Ebre is a natural bay model for Marteilia spp. (Paramyxea) dynamics and life-cycle studies.

Marteilia spp. are paramyxean parasites that affect several bivalve species of economic interest, such as Ostrea edulis and Mytilus galloprovincialis. Certain aspects of Marteilia spp., such as their life cycle and host affinity and infection dynamics, still remain unknown. The 'Delta de l'Ebre' constitutes a natural model for the study of the life cycle of the parasite Marteilia, since uninfected mussels and flat oysters immersed in the bays can become infected. This, along with the geographical and ecological characteristics of the bays, make it a very interesting location to study the Marteilia life cycle. Preliminary results concerning marteiliosis, mainly in mussels, such as prevalence dynamics, infectious periods, host affinity and host intermediate candidates are reported in the present paper. This information will be required for further, more exhaustive, studies in the bays of the Ebre delta.

Dynamics of the parasite Marteilia refringens (Paramyxea) in Mytilus galloprovincialis and zooplankton populations in Alfacs Bay (Catalonia, Spain).

Since the first description of Marteilia refringens (Paramyxea) in flat oysters Ostrea edulis in 1968 in the Aber Wrach, Brittany (France), the life-cycle of this parasite has remained unknown. However, recent studies, conducted in the 'claire' system, have proposed the planktonic copepod Acartia grani as a potential intermediate host for the parasite. Nevertheless, experimental transmission of the parasite through the copepod has failed. Recent studies in this field have reported the presence of the parasite in zooplankton from the bays of the Delta de l'Ebre, a more complex and natural estuarine environment than that of the claire. As a result, 2 new Marteilia host species were proposed: the copepods Oithona sp. (Cyclopoida) and an indeterminate Harpaticoida. Consequently, the objective of the present work was to study the dynamics of Marteilia in the zooplankton community from one of the bays, Alfacs Bay, as well as the dynamics of the parasite in cultivated mussels during 1 complete year. Six different zooplankton taxa appeared to be parasitized by M. refringens, including copepods (3 Calanoida, Acartia discaudata, A. clausi and A. italica; 1 Cyclopoida, Oithona sp.; and 1 Harpacticoida, Euterpina acutifrons), and larval stages of decapod crustaceans (zoea larvae of Brachyura, probably Portumnus sp.). These taxa are thus proposed as new subjects for study, since they could be intermediate hosts in the infection process of mussels by Marteilia.

Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom.

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

Culture media for the differentiation of isolates of Yersinia ruckeri, based on detection of a virulence factor.

Strains of the bacterial fish pathogen Yersinia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1% SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri.

Virulence of Yersinia ruckeri serotype I strains is associated with a heat sensitive factor (HSF) in cell extracts.

Cell extracts of Yersinia ruckeri (serotype I) were examined by SDS-PAGE and Western blotting. An unusual band, termed heat-sensitive factor (HSF) was observed in extracts of virulent strains only. It is thought to be lipid in nature; no differences could be detected in the region of the band in protein profiles of virulent and avirulent strains. When trout were infected either by intraperitoneal injection or bath immersion, mortalities occurred only with HSF+ strains. The HSF appears to be an important virulence determinant of Y. ruckeri.