Beatriz Magariños

Effect of oral administration of glucans on the resistance of gilthead seabream to pasteurellosis

Abstract The present study evaluated the effect of oral administration of three types of glucan on the resistance of gilthead seabream to Photobacterium damselae subsp. piscicida and on the activity of its phagocytes. Groups of fish were fed with a basal diet (controls) or with the basal diet supplemented with glucan for two different periods of time before being bath challenged with the bacterium. Groups of fish fed with 1 and 10 g kg −1 of glucan for a short period (2 weeks with glucan and 1 week with the basal diet) showed a higher degree of protection against pasteurellosis than the control group. This was especially pronounced in groups of fish fed with the higher concentrations of glucan. The respiratory burst and phagocytic activity of spleen phagocytes varied with time but no significant differences were found between groups in the third week when the challenge was carried out. The group of fish fed with 1 g kg −1 of glucan for longer periods of time (2 weeks with glucan, 1 week with the basal diet, 2 weeks with glucan, and 1 week with the basal diet) also showed enhanced protection against P. damselae . However, the group fed with 5 g kg −1 had mortality rates comparable to the control group, and protection in the group fed with 10 g kg −1 of glucan was significantly lower than in the control group. These results suggest that glucans can be used in the diet to prevent or reduce mortalities in gilthead seabream due to pasteurellosis, and they show the importance of the concentration and the period of administration of glucan to obtain optimal protection against this disease.

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.

Acylhomoserine lactone production and degradation by the fish pathogen Tenacibaculum maritimum, a member of the Cytophaga–Flavobacterium–Bacteroides (CFB) group

Tenacibaculum maritimum (formerly Flexibacter maritimus) is a filamentous, biofilm-forming member of the Cytophaga-Flavobacterium-Bacteroides group (or Bacteroidetes), which causes the widely distributed marine fish disease tenacibaculosis. A search for N-acylhomoserine lactones (AHLs) quorum-sensing (QS) signals in the culture media of nine representative strains of this species using different biosensor strains revealed the presence of short-type AHL activity in all of them. N-butyryl-L-homoserine lactone (C4-HSL) was identified in T. maritimum NCIMB2154(T) by LC-MS. A degradation activity for long-acyl AHLs (C10-HSL) was subsequently demonstrated in T. maritimum NCIMB2154(T). The acidification of the culture medium after degradation did not allow the recovery of C10-HSL, which indicates a possible acylase-type degradation activity. Even though the physiological processes under the control of AHL-mediated QS in T. maritimum need to be further characterized, this discovery extends the paradigm of AHL-mediated QS signalling beyond the Proteobacteria and reinforces its ecological significance.

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.

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens

BackgroundThe turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis.ResultsA total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot.ConclusionA collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

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).

Usefulness of the API-20E system for the identification of bacterial fish pathogens.

Abstract A total of 223 isolates belonging to motile Aeromonas, A. salmonicida, Vibrio anguillarum, Pasteurella piscicida and Yersinia ruckeri species were tested by the API-20E system and the results compared with those obtained with standard biochemical tube and plate tests. Depending on the species, the API-20E yielded false negative and positive reactions for fermentation of different sugars, lysine decarboxylase (LDC), Voges Proskauer (VP), citrate and gelatinase reactions. Thirteen of 32 (41%) A. salmonicida strains and 9 of 53 (17%) Y. ruckeri isolates were correctly identified using the API index. Forty-five of 69 (65%) motile Aeromonas tested (including 34 A. hydrophila, 10 A. sobria and 1 A. caviae strains) were identified as A. hydrophila. The low discrimination profiles generated by 9 Yersinia and 2 motile Aeromonas isolates were avoided using the supplementary tests cited in the API index. In the case of V. anguillarum and P. piscicida, which are not at present included in the API-20E system, 35 of 53 strains of V. anguillarum were wrongly identified as A. hydrophila, and all the P. piscicida isolates were incorrectly identified as Pseudomonas fluorescens/Ps. putida. A large number of isolates, including some reference strains, produced uncoded profiles. From these results, we consider that the API-20E system will be a useful tool for field diagnosis of bacterial fish diseases if its database is expanded to include V. anguillarum, P. piscicida, A. sobria and A. caviae species and the new profiles generated by strains of species already considered. In addition, some reactions necessary to discriminate between strains which share the same profile number are proposed.

Vaccination trials on gilthead seabream (Sparus aurata) against Pasteurella piscicida

A comparative study of the efficacy of two vaccine formulations, a whole-cell bacterin (WCB) and a toxoid-enriched whole-cell vaccine (WCEB), against Pasteurella piscicida was conducted by bath immersion in gilthead seabream in order to evaluate the role of the extracellular products (ECP) as protective antigens against this pathogen. With this aim, two strains showing differences in their ECP composition were used to prepare both vaccines. Only the toxoid-enriched vaccine conferred protection against P. piscicida within a 4-week period. The relative percent survival (RPS) acheived with this type of vaccine ranged between 37 and 41 depending on the bacterial strain and dose used in the challenge. Although this protection level is not very high, we consider that it is valuable considering the economic importance of the fish susceptible to pasteurellosis throughout the world. The booster immunization with the WCEB P. piscicida formulation did not increase the protection levels of gilthead seabream. The antibody response in the sera of both immunized fish groups was very low with no correlation between the level of agglutinating antibodies and the protection. In addition, this vaccine did not confer cross-protection against serotypes 01 and 02 of Vibrio anguillarum.

Development of an effective Edwardsiella tarda vaccine for cultured turbot (Scophthalmus maximus).

Since 2004 Edwardsiella tarda has become one of the most important emerging pathogens in turbot aquaculture industry in Europe causing serious economic losses. Therefore, this study aimed to design an effective vaccination strategy to prevent edwardsiellosis in this fish species. Two vaccine formulations, an adjuvanted vaccine and an aqueous bacterin, and different routes of administration, bath and intraperitoneal injection (i.p.), were tested. The effectiveness of the different immunization strategies was evaluated in terms of relative percent survival (RPS) and antibody levels. On the basis of the results obtained we recommend the i.p. administration of a non-mineral oil adjuvanted vaccine via i.p., which confers RPS values over 90% at least 6 months post-vaccination.

Molecular Fingerprinting of Fish-Pathogenic Lactococcus garvieae Strains by Random Amplified Polymorphic DNA Analysis

ABSTRACT In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Lactococcus garvieae, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of L. garvieae studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.