Jesús L. Romalde

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.

Phenotypic and pathobiological characteristics of Pasteurella piscicida

Abstract Pasteurellosis, caused by Pasteurella piscicida , is one of the most threatening diseases of wild and cultured marine fish, and has been reported from many geographical areas including the USA, Japan and the Mediterranean countries. The objective of this article is to construct a picture of the current state of knowledge about this bacterial pathogen and the pathogenesis of the disease it causes. We review some important questions such as the controversial taxonomic position of the bacterium, and its main virulence mechanisms. The epidemiology of the disease, the routes of transmission and the putative reservoirs of P. piscicida in the environment are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, including new diagnostic procedures, chemotherapy, employment of immunostimulants, and improvements in immunization programs.

Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs.

Shellfish production is seriously affected by bacterial pathogens that cause high losses in hatcheries and in the aquaculture sector. A number of Vibrio species are considered important pathogens and have provoked severe mortality outbreaks. The pathologies caused by vibrios in bivalves have been described since the 1960s; however, over recent years, successive episodes of high mortality have been recorded due to these microorganisms. The present work provides an updated overview of the different studies performed in relation with the diversity of Vibrio spp. associated to bivalves. Special attention is given to the main Vibrio diseases and implicated species affecting the different life stages of cultured bivalves.

Efficacy of intraperitoneal and immersion vaccination against Enterococcus sp. infection in turbot

In this study the effectiveness of a toxoid-enriched whole-cell bacterin (ET-2) in cultured turbot (Scophthalmus maximus) against Enterococcus sp. was evaluated by bath immersion and intraperitoneal (i.p.) injection. In addition, the influences of fish size as well as enrichment of the standard semimoist diet with the commercial preparation Trouvitol plusR, which contains β-glucan from yeast, were also investigated. Regardless of fish size, type of diet, and bacterial dose employed in the experimental challenges, our Enterococcus bacterin ET-2 proved to be very effective when it was delivered by injection. The RPS (relative percent of survival) values achieved by this route ranged from 89 to 100 (for 45-g turbot) and from 67 to 86 (for 150-g turbot), depending on the bacterial levels and time of experimental challenge. Moreover, this strong degree of protection lasted for at least 1 year. No circulating antibodies were detected in the vaccinated turbot. However, the injected bacterin, irrespective of the diet utilized, elicited a significant (P < 0.01) increase of the phagocytic activity in the spleen. Although the glucan alone also induced an enhancement of the non-specific defence mechanisms, this stimulation was not correlated with the protection of turbot against Enterococcus infection.

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.

Aeromonas piscicola sp. nov., isolated from diseased fish.

Four Aeromonas strains (S1.2(T), EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA-DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2(T) (=CECT 7443(T), =LMG 24783(T)) as the type strain.

Antigenic and Molecular Characterization of Yersinia ruckeri Proposal for a New Intraspecies Classification

Summary The serological and molecular properties of a group of 17 Yersinia ruckeri strains isolated from cultured rainbow trout in Spain were compared with a total of 36 collection strains isolated from fish in other geographic areas. The serological relationship among isolates, as well as the study of their antigenic determinants (LPS and membrane proteins) allow to propose a new serotyping scheme with four O-serotypes (from O1 to O4). Moreover, serotypes O1 and O2 were divided in two and three groups respectively based mainly on minor differences observed in LPS and membrane protein profiles. The patterns obtained in the analysis of the LPS and proteins present in the extracellular products also support the serological scheme proposed. The strains showed a great genetic homogeneity, displaying similar DNA fingerprint patterns regardless of their serotype. In contrast, only the serotype O1 isolates harbour a plasmid of about 50 MDa, while the strains of the other serotypes presented no extrachromosomical DNA or only small plasmids (less than 10 MDa).

Microflora associated with healthy and diseased turbot (Scophthalmus maximus) from three farms in northwest Spain

A comparative analysis of the microbiological quality of three turbot (Scophthalmus maximus) farms (A, B, and C) located in Galicia (northwest Spain) is given. The microbial load and types of bacteria in the internal organs (liver and kidney) of apparently healthy fish was monitored over a year, and all the disease problems occurring during this survey were analyzed. The percentage of healthy turbot in which positive bacterial growth was obtained was relatively high in the three ongrowing facilities. Farm A exhibited the poorest conditions of fish health with an average of 42% fish infected, while farm B showed the best microbiological quality with 27% of turbot harbouring bacteria in the internal organs. In all three farms, a wide range of bacteria was found in healthy turbot with Vibrio (V. splendidus-V. pelagius, V.fisheri-V. harveyi and Vibrio spp.) and Pseudomonas spp. being the predominant groups comprising at least 80% of the total bacterial isolates in each farm. The highest number of pathological problems (22) with the most diverse bacterial flora occurred in farm A. Vibrio spp. and Pseudomonas spp. were the most prevalent bacteria recovered from diseased turbot. Haemorrhages in palate and jaws, tail and fins, and ulcerative lesions were the most frequent external clinical sings of diseased fish recorded in the three farms. However, it was not possible to associate a particular bacterial species with a specific pathology. Routine use in farm A of oxolinic acid and nitrofurantoin may have led to the development in the Vibrio strains of resistances to both chemotherapeutants (up to 25%).

Review of probiotics for use in bivalve hatcheries.

The aquaculture of bivalve molluscs has attained a considerable level of production but it is not enough to cover the demand of worldwide consumers. In the development of this sector, hatcheries play an important role, as suppliers of competent spat of different bivalves, including species with an aquaculture based on natural extraction present. Besides, these installations may help in the recovery of exhausted natural beds and in the obtaining of populations under genetic selection. Unfortunately, the disease outbreaks caused by bacterial pathogens are frequent, with the loss of complete batches, compromising the regular production and the economic viability of the industry. There are many descriptive studies about these outbreaks, but only a few focused on the control of microbiota. The particularities of bivalve aquaculture in hatchery must be taken into account to design methods of control. A common environment is shared by larvae and bacteria, including both beneficial and potentially pathogenic. The filter-feeding behaviour of larvae increases the strong influence of these populations. The classical treatments are directed toward to the complete elimination of bacteria from culture seawater. That objective is unfeasible, because the cultures are not axenic, and undesirable, since some bacteria enhance larval development. Taking into account these considerations, the most promising alternative is the use of probiotic bacteria. In this review we summarize the scientific literature about this subject, considering the particularities of bivalve larval cultures and the need to adapt the concept of probiotic and the strategies to use in marine bivalve hatcheries.