An overview of 20 years of studies on the prevalence of human enteric viruses in shellfish from Galicia, Spain.

Galicia (NW Spain) has 1490 km of coastline, and its particular topography, characterized by the presence of fiord-like inlets, called rías, with an important primary production, makes this region very favourable for shellfish growth and culture. In fact, Galicia is one of the most important mussel producers in the world. Due to its proximity to cities and villages and the anthropogenic activities in these estuaries, and despite the routine official controls on the bivalve harvesting areas, contamination with material of faecal origin is sometimes possible but, current regulation based on Escherichia coli as an indicator micro-organism has been revealed as useful for bacterial contaminants, this is not the case for enteric viruses. The aim of this review is to offer a picture on the situation of different harvesting areas in Galicia, from a virological standpoint. A recompilation of results obtained in the last 20 years is presented, including not only the data for the well-known agents norovirus (NoV) and hepatitis A virus (HAV) but also data on emerging viral hazards, including sapovirus (SaV), hepatitis E virus (HEV) and aichivirus (AiV). Epidemiological differences related to diverse characteristics of the harvesting areas, viral genotype distribution or epidemiological links between environmental and clinical strains will also be presented and discussed. The presentation of these historical data all together could be useful for future decisions by competent authorities for a better management of shellfish growing areas.

Mortality event involving larvae of the carpet shell clam Ruditapes decussatus in a hatchery: isolation of the pathogen Vibrio tubiashii subsp. europaeus.

Diseases caused by bacteria belonging to the genus Vibrio are a common, as yet unresolved, cause of mortality in shellfish hatcheries. In this study, we report the results of routine microbiological monitoring of larval cultures of the carpet shell clam Ruditapes decussatus in a hatchery in Galicia (NW Spain). Previous episodes of mortality with signs similar to those of vibriosis affecting other species in the installation indicated the possibility of bacterial infection and led to division of the culture at the early D-veliger larval stage. One batch was cultured under routine conditions, and the other was experimentally treated with antibiotic (chloramphenicol). Differences in larval survival were assessed, and culturable bacterial population in clams and sea water was evaluated, with particular attention given to vibrios. Severe mortalities were recorded from the first stages of culture onwards. The pathogen Vibrio tubiashii subsp. europaeus was detected in both batches, mainly associated with larvae. Moreover, initial detection of the pathogen in the eggs suggested the vertical transmission from broodstock as a possible source. Experimental use of antibiotic reduced the presence and diversity of vibrios in sea water, but proved inefficient in controlling vibrios associated with larvae from early stages and it did not stop mortalities.

Pharmacokinetic model of florfenicol in turbot (Scophthalmus maximus): establishment of optimal dosage and administration in medicated feed.

The pharmacokinetics of florfenicol (FF) in turbot (Scophthalmus maximus) was studied after single intravenous (10 mg kg-1 ) and oral (100 mg kg-1 ) administration. The plasma concentration-time data of florfenicol were described by an open one-compartment model. The elimination half-life (t1/2 ) was estimated to be 21.0 h, and the total body clearance, Cl, was determined as 0.028 L kg h-1 . The apparent volume distribution (Vd ) was calculated to be 0.86 L kg-1 and the mean residence time (MRTiv ) was 30.2 h. Following oral administration, the maximum plasma concentration (Cmax ) of 55.4 µg mL-1 was reached at 12 h (Tmax ). The absorption constant (ka ) was 0.158 h-1 . The bioavailability was estimated to be 57.1%. The low bioavailability observed at higher doses was explained by the saturation of the mechanisms of absorption. The drug absorption process was limited by its inherent low solubility, which limited the amount of available FF absorbed in the gastrointestinal tract. Based on the pharmacokinetic data, an optimal dosing schedule for FF administration is hereby provided. Based on the minimum inhibitory concentration found for susceptible strains of Aeromonas salmonicida, oral FF administration of first, an initial dose of 30 mg FF kg-1 , followed by 6 maintenance doses at 18 mg kg-1 /daily could be effective against furunculosis in turbot.

Phytoplankton production systems in a shellfish hatchery: variations of the bacterial load and diversity of vibrios.

AIMS: Outbreaks of disease caused by some Vibrio species represent the main production bottleneck in shellfish hatcheries. Although the phytoplankton used as food is one of the main sources of bacteria, studies of the associated bacterial populations, specifically vibrios, are scarce. The aim of the study was the microbiological monitoring of the microalgae as the first step in assessing the risk disease for bivalve cultures. METHODS AND RESULTS: Two phytoplankton production systems were sampled weekly throughout 1-year period in a bivalve hatchery. Quantitative analysis revealed high levels of marine heterotrophic bacteria in both systems throughout the study. Presumptive vibrios were detected occasionally and at low concentrations. In most of the cases, they belonged to the Splendidus and Harveyi clades. CONCLUSIONS: The early detection of vibrios in the microalgae may be the key for a successful bivalve culture. Their abundance and diversity were affected by factors related to the hatchery environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents the first long study where the presence of vibrios was evaluated rigorously in phytoplankton production systems and provides a suitable microbiological protocol to control and guarantee the quality of the algal cultures to avoid the risk of transferring potential pathogens to shellfish larvae and/or broodstock.

Vibrios in hatchery cultures of the razor clam, Solen marginatus (Pulteney).

Hatchery culture of the razor clam, Solen marginatus (Pulteney), has recently been developed in Galicia (NW Spain). However, recurrent episodes of mortalities of larval and post-larval cultures have been recorded during the course of various studies. The disease signs were similar to those described for other bivalve species in outbreaks caused by bacteria of the genus Vibrio. In this article, we present the results of microbiological monitoring of two batches of razor clams with different survival rates. All fermentative isolates were identified as members of the Splendidus clade within the genus Vibrio. Some of these isolates, identified as Vibrio splendidus-like, were clearly associated with the batch suffering mortalities, indicating their possible role as pathogens. Similar strains were found in the broodstock, suggesting vertical transmission of these bacteria. This is the first study of the microbiota associated with hatchery culture of S. marginatus, and the results will provide useful information for the optimization of a protocol for hatchery culture of this bivalve species.

Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish.

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.

Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot.

Edwardsiella tarda is an enterobacterial fish pathogen that causes mortality in various fish species worldwide. In this study, we analyzed the intraspecific variability in a collection of E. tarda strains isolated from turbot. To do this we employed 4 polymerase chain reaction (PCR)-based methods: (1) random amplified polymorphic DNA (RAPD), (2) enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), (3) repetitive extragenic palindromic-PCR (REP-PCR) and (4) BOX-PCR. E. tarda isolates from different hosts were also included for comparison. E. tarda strains from turbot showed high molecular homogeneity when RAPD (primers P3 and P6), ERIC-PCR and BOX-PCR were employed. However, with regard to the REP-PCR and RAPD (primers P4 and P5) techniques, different genetic groups could be established within these isolates using either technique. The 2 RAPD types presented an 85% similarity, while those obtained with REP-PCR showed 74% similarity. Based on the results obtained, although a high genetic homogeneity was found in turbot isolates, the RAPD test (with primers P4 and P5) and REP-PCR were capable of discrimination within these strains, and they are therefore considered the most appropriate typing methods for studies of edwardsiellosis in turbot.

Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment.

Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.

Development of a rapid, sensitive and non-lethal diagnostic assay for the detection of viral haemorrhagic septicaemia virus.

A non-lethal diagnostic procedure based on polymerase chain reaction (PCR) technology was developed to detect viral haemorrhagic septicaemia virus (VHSV). Sensitivity of the assay was tested using purified viral RNA and seeded tissues. Detection limits of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay were estimated to be 10 fg of purified RNA and 0.97 x 10(3) or 10(0) TCID(50)/g of seeded tissue, depending on the experimental approach employed (viral adsorption allowed for 1 or 24h). Addition of nested PCR increased sensitivity up to 100-fold when cDNA excised from the agarose gel was used as template. Both, RT-PCR and nested RT-PCR, as well as Southern blot were applied to RNA extracted from blood of experimentally infected brown trout and the results were compared with those obtained by applying the same techniques to tissues and also with those of conventional viral isolation in cell culture. The superiority of the nested RT-PCR applied to blood samples has been clearly demonstrated in terms of sensitivity, obtaining positive results in 85% of fish tested, as against 40% obtained by RT-PCR and Southern blot, and only 5% viral isolations in cell culture. This procedure could turn into an important tool for screening of wild stocks as well as valuable individuals in commercial fish farms, since it makes to kill the fish unnecessary.

Genetic transformation of Vibrio anguillarum and Pasteurella piscicida by electroporation.

Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 x 10(3) transformants per micrograms DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 x 10(2) transformants per micrograms pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.

Influence of the growth conditions on the hydrophobicity of Renibacterium salmoninarum evaluated by different methods.

The influence of medium and salinity on the cell surface hydrophobicity of Renibacterium salmoninarum was investigated using three different methods: bacterial adherence to hydrocarbons (BATH), salt agglutination test (SAT), and binding to nitrocellulose filters (NCF). The possible relationship among hydrophobicity, haemagglutination and adherence to cell lines was also evaluated. R. salmoninarum showed to be highly hydrophobic regardless of the growth conditions or technique employed. Nevertheless, slight differences can be detected depending on the method used. In the SAT and NCF assays very uniform values were obtained within the strains. All the R. salmoninarum isolates agglutinated in (NH4)2SO4 in a range of 0.05-0.2 M and displayed a 77-100% of adherence to nitrocellulose filters. However, more variable results were observed in the BATH method depending on the hydrocarbon, buffer and strain employed. Although all of the isolates produced haemagglutinins for homeotherm erythrocytes, the majority of them failed to agglutinate poikilothermic red blood cells and were unable to adhere to fish cell lines. Therefore, a general correlation among hydrophobicity, agglutinating capacity for fish erythrocytes and adherence to fish cells can not be established for R. salmoninarum.

Adherence and invasive capacities of the fish pathogen Pasteurella piscicida.

Pasteurella piscicida strains were weakly or moderately adherent to cell lines, the levels of attachment being variable depending on the cells employed. All the isolates exhibited the highest binding capacity to CHSE-214 cells. Adhesive capacities were affected by heat and sugars but not by proteinase K or by treatment with antisera raised against the lipopolysaccharides of P. piscicida, implicating components of glycoprotein(s) as ligands in the adhesion process. The isolates showed a great binding capacity to intestines from the marine fish hosts gilthead sea bream, sea bass and turbot, with values ranging from 10(4) to 10(5) bacteria/g. Although the P. piscicida strains showed a weak invasiveness in the poikilothermic cell lines employed as in vitro model, the bacteria remained viable inside the infected cells at least for 2 days. The invasion process was inhibited by cytochalasin D indicating the active participation of the host cytoskeleton in the internalization of P. piscicida.

The detection of two antigenic groups among Renibacterium salmoninarum isolates.

The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected.

Genetic analysis of turbot pathogenic Streptococcus parauberis strains by ribotyping and random amplified polymorphic DNA.

Ribotyping and RAPD profiling of a collection of 18 Streptococcus parauberis strains isolated from diseased turbot in Galicia (NW Spain) was performed in order to analyze the possible genetic variability within this bacterial fish pathogen. In addition, the value of this technique for intraspecific classification and epidemiological studies was evaluated. Ribopatterns of DNA digested with three endonucleases and hybridized with a cDNA probe complementary to highly conserved sequences in the 16S and 23S rRNA genes showed a great homogeneity among the turbot isolates. Compared with ribotyping, RAPD appeared to be a reliable and fast technique for discriminating between isolates of S. parauberis on the basis of their farm of isolation and, therefore, represents a powerful tool for epidemiological studies of this fish pathogen.

Plasmids and factors associated with virulence in environmental isolates of Vibrio cholerae non-O1 in Bangladesh.

Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-O1 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7-115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxic strains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-O1 strains. Conjugal transfer assays demonstrated that the 115-Mda plasmid harboured by some of the toxigenic V. cholerae non-O1 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mol.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-O1 strains. Regardless of their pathogenic potential, V. cholerae non-O1 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced.

Plasmid associated virulence properties of environmental isolates of Aeromonas hydrophila.

The plasmid profiles, and their association with antimicrobial resistance, of 60 strains of Aeromonas hydrophila isolated from fish, shellfish and water were investigated. Only two strains were susceptible to all the antimicrobial agents tested; the highest incidences of resistance were to tetracycline (96.7%), prystanamycin (93.3%), ampicillin (91.7%) and cephalothin (91.7%). Forty strains harboured one or more plasmids and the plasmid profile most frequently detected (15%) was the association of three small plasmids of 4.2, 3.2 and 2.8 Mda. Curing experiments indicated that the plasmid-free derivative strains simultaneously lost their resistance determinants to tobramycin, neomycin, gentamicin and kanamycin. More than 90% of the strains tested produced siderophores and displayed haemolytic activity. However, the relationship between these virulence characters and the presence of plasmids was different; in 74.5% of the strains there was siderophore production and plasmids were detectable, whereas only 60% of the strains simultaneously possessed plasmids and haemolytic activity.

Prevalence of enterovirus and hepatitis A virus in bivalve molluscs from Galicia (NW Spain): inadequacy of the EU standards of microbiological quality.

A study of the presence of hepatitis A virus (HAV) and enterovirus (EV) in shellfish from the northwestern coast of Spain, one of the most important mussel producers in the world, was carried out employing dot-blot hybridization and RT-PCR techniques. In addition, bacterial contamination of the samples was evaluated by Escherichia coli (EC) counts, according to the European Union (EU) standards of shellfish microbiological quality. Shellfish samples included raft-cultured and wild mussels, as well as wild clams and cockles. Bacterial counts showed that the majority of samples (40.8%) could be classified as moderately polluted following the EU standards, and therefore should undergo depuration processes. However, differences in bacterial contamination were observed between cultured mussel and wild shellfish. Thus, percentage of clean samples (<230 EC/100 g shellfish) was clearly higher in cultured mussels (49.1%) than in wild mussels (22.8%) or clams and cockles (10.7%). HAV was detected in 27.4% and EV in 43.9% of the samples that were analyzed. Simultaneous detection of both viral types occurred in 14.1% of the samples. Statistical tests of dependence (chi-square test) showed no relationship either between viral and bacterial contamination, or between the presence of HAV and EV. Comparative analysis of hybridization and RT-PCR for viral detection yielded different results depending on the virus type that was studied, RT-PCR being effective for HAV but not for EV detection. The obtained results reinforce once again the inadequacy of bacteriological standards to assess viral contamination and suggest that although virological analysis of shellfish is possible by molecular techniques, interlaboratory standardization and validation studies are needed before the routine use in monitoring shellfish microbiological safety.

The humoral immune response of turbot to recently isolated pathogenic Enterococcus strains. Cross-reactivity with other Gram-positive bacteria.

An Enterococcus sp. causing severe mortalities among farmed turbot (Scophthalmus maximus) has recently been detected in northwest Spain. We found that specific turbot serum antibodies raised against isolate RA-99.1 of the new pathogen by intraperitoneal immunization, did not cross react in enzyme-linked immunosorbent assay (ELISA) with other enterococcal or non-enterococcal Gram-positive bacteria. In immunoblotting, antibodies raised against strain RA-99.1 recognized the same components in the homologous total soluble antigen preparation (TSA) as in TSA of two other isolates of the pathogen obtained from different farms. Anti-RA-99.1 serum also recognized some components of the TSA of several other Gram-positive bacteria. Immunogold labelling indicated that the antigens which provoke the humoral immune response to this pathogen are located mainly on the bacterial surface.

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae.

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.

Isolation of viral hemorrhagic septicemia virus from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap.

Viral hemorrhagic septicemia virus (VHSV) was isolated from apparently healthy Greenland halibut Reinhardtius hippoglossoides caught in the Flemish Cap, a deep fishing ground in the North Atlantic Ocean in international waters near Newfoundland. The identity of the virus was confirmed by electron microscopy, immunodot, seroneutralization and reverse transcriptasepolymerase chain reaction. In the serology assays, all isolates reacted in the immunodot assay with a polyclonal antiserum against the European VHSV Type Strain F1, and were neutralized by the same antiserum, although most of the strains showed low or moderate neutralization titers. None of the isolates were detected by immunofluorescence using a specific monoclonal antibody against a nucleocapsid-related protein of VHSV F1. This is the first report of VHSV isolated from wild Greenland halibut, which represents a new host species for the virus, and it is also the first evidence of VHSV in a location close to the Atlantic coast of North America. This isolation indicates that VHSV is more widely distributed than has been thought, and appears to support a marine origin of this virus.