A challenge model for Tenacibaculum maritimum infection in turbot, Scophthalmus maximus (L.).
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Animals↗
Biomedical subjects
B Magariños
Publications and source records attributed to B Magariños.
Characterization of Edwardsiella tarda strains isolated from turbot, Psetta maxima (L.).
The biochemical, serological and molecular characteristics of a group of 21 Edwardsiella tarda strains isolated from turbot, Psetta maxima, in two different areas of Europe were analysed and compared with a total of 13 strains of this bacterial species with different geographical and host origins. All the turbot isolates were biochemically identical to the E. tarda strains included as reference. The use of different techniques including microagglutination, dot blot and Western blot of lipopolysaccharides allowed us to determine that all the turbot isolates constitute an homogeneous and distinctive serological group. Genetic analysis by randomly amplified polymorphic DNA (RAPD) analysis demonstrated that although the E. tarda strains from turbot were compiled in a unique group using the primers P3 and P6, two clonal lineages could be detected when oligonucleotides P4 and P5 were employed.
Agglutination Tests↗
Effectiveness of a divalent vaccine for sole, Solea senegalensis (Kaup), against Vibrio harveyi and Photobacterium damselae subsp. piscicida.
The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly.
Animals↗
Vaccination strategies to prevent emerging diseases for Spanish aquaculture.
In recent years, three serious diseases have emerged in Spanish aquaculture. These are lactococcosis caused by Lactococcus garvieae, which is of economical importance in rainbow trout (Oncorhynchus mykiss); pseudomonadiasis caused by Pseudomonas anguilliseptica which affects gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus); and flexibacteriosis caused by Tenacibaculum maritimum which became a devastating problem in the emerging culture of sole (Solea spp). To obtain useful information for the design and development of new vaccines, antigenic characterisation of representative strains was performed. In this work we present the strategies adopted for the vaccine formulation (strains included, use of adjuvants) and administration (route, necessity of booster, etc.). The results from laboratory and/or field vaccination trials performed showed that for lactococcosis, protection lasting for five months was obtained with an oil-adjuvanted bacterin formulation. Unadjuvanted bacterin gave only a short duration of protection, which could, however, be prolonged by an antigen boost administered via the feed. A bacterin against Pseudomonas anguilliseptica gave protection for 12 weeks when tested in an experimental challenge trial in turbot. Besides the flexibacteriosis vaccine developed by our group for turbot, and due to the antigenic host-associated variability within T. maritimum, a new bacterin was developed against this bacterium to be used specifically in sole. This new bacterin, administered to sole by intraperitoneal injection, yielded RPS values of 94 % six weeks after immunization. In conclusion, these results suggest that vaccination constitutes a cost-effective method of controlling diseases that have emerged in the most important fish species being cultured in Spain.
Adjuvants, Immunologic↗
Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR.
AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.
Animals↗
Dormancy as a survival strategy of the fish pathogen Streptococcus parauberis in the marine environment.
The fate of Streptococcus parauberis in seawater and sediment microcosms at different temperatures (6 and 22 degrees C) was investigated by comparing the survival dynamics of 2 strains of this bacterial species, isolated respectively from diseased turbot and cattle. The turbot and the bovine isolate showed similar survival kinetics, remaining culturable for approximately 1 mo in water and 6 mo in sediment. A slight influence of temperature on the stability of the cells was observed, in that the number of culturable cells was about 1 log10 unit higher at 6 than at 22 degrees C. During the starvation period, the metabolic activity of the cells, after suffering a strong reduction during the first 12 d, stabilized at levels ranging from 20 to 40% of the initial values. However, in all the microcosms, the acridine orange (AO) and 4',6-diamidino-2-phenilindole (DAPI) counts remained at about 10(5) cells ml(-1) throughout the experimental period, even when cells became undetectable by standard plate count methods. The addition of fresh medium to microcosms containing nonculturable cells induced the return to culturability of S. parauberis strains. On the basis of these results, it seems that S. parauberis has the ability to enter into a viable but nonculturable (VBNC) state. Dormant cells of the turbot isolate maintained their infectivity and pathogenic potential for fish.
Animals↗
Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis.
In this work, we applied the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida), an important pathogen for different marine fish. Regardless of the oligonucleotide primer employed, the 29 isolates of Ph. damselae subsp. piscicida tested were separated into two groups, the RAPD-PCR analysis differentiated the European strains from the Japanese strains. The similarity between both groups estimated on the basis of the Dice coefficient was 75-80%. These results show that European and Japanese isolates of Ph. damselae subsp. piscicida, regardless of their host fish species, belong to two different clonal lineages. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.
Animals↗
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.
Animals↗
Assessment of a magnetic bead-EIA based kit for rapid diagnosis of fish pasteurellosis.
The accuracy of the magnetic beads-EIA based BIONOR AQUAEIA-Pp kit for the rapid diagnosis of pasteurellosis was evaluated. The kit reacted with all the Photobacterium damselae subsp. piscicida strains included in this study, with a detection limit of 10(4) bacteria/ml. However, non-specific reactions were observed with isolates of Ph. damselae subsp. damselae or Ph. histaminum when the bacterial concentration was high (10(9)-10(10) bacteria/ml). Similar findings in specificity and sensitivity were observed when the kit was applied to experimentally infected fish tissues. However, since those bacterial species are not usually found in the fish species susceptible to pasteurellosis, the AQUAEIA kit appears applicable for a rapid screening of the disease. In addition, when the kit was utilized to analyze cultured populations of seabream, it allowed the detection of the pathogen, not only in individuals affected by the disease, but also in asymptomatic carrier fish. Furthermore, the positive detection of Ph. damselae subsp. piscicida in broodstock gonads, seminal, and ovaric fluids, and also in eggs indicated the possibility of vertical transmission of pasteurellosis.
Animals↗
Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.
A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.
Bacterial Typing Techniques↗
Immunization with bacterial antigens: pasteurellosis.
Pasteurella piscicida is the aetiological agent of pasteurellosis or pseudotuberculosis, one of the most threatening diseases of wild and cultured marine fish. This bacterium has been reported from many geographical areas including USA, Japan, and the Mediterranean countries. In this review, the biochemical, serological, and molecular characteristics of the pathogen are described. In addition, its main virulence mechanisms, such as the presence of capsule, the iron uptake system, and the phospholipase activity, as well as their putative role in the pathogenicity of P. piscicida are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, with a special emphasis on the vaccination programmes and the most effective protective antigens to be included in the vaccine formulations.
Adjuvants, Immunologic↗
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.
Animals↗
Influence of the capsular layer on the virulence of Pasteurella piscicida for fish.
The influence of the capsule of Pasteurella piscicida on the cell surface properties of this microorganism as well as on the virulence and the capacity of the strains to grow in fish sera was examined. Although all the P. piscicida strains synthetized an additional exostructure in glucose-enriched-medium, only virulent strains constitutively synthetized capsule. The cell surface of all the P. piscicida isolates showed a low hydrophobic nature. No strains pelleted in broth culture (SP-) and all of them were stable after boiling (PAB). All isolates attached to the fish cell line CHSE-214 with values of adherence ranging from 2 to 5% of the initial bacterial inoculum. The presence of induced capsular material caused changes in some cell surface characteristics such as hydrophobicity and stability after boiling. A decrease in the adherence capacity of all the P. piscicida strains was also observed. However, the capsule increased the degree of virulence for fish of the nonpathogenic strains (LD50 was reduced in about 4 log) and conferred to all the isolates resistance to serum killing. Therefore, these results indicate that the presence of capsule can play an important role in the pathogenesis of P. piscicida.
Animals↗
Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.
Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.
Animals↗
Fatty acid profiles of "Pasteurella" piscicida: comparison with other fish pathogenic gram-negative bacteria.
The fatty acid methyl ester (FAME) profiles of "Pasteurella" piscicida were determined by gas chromatography and subjected to numerical analysis in comparison with those obtained for Vibrio anguillarum, Aeromonas salmonicida and Pasteurella species of clinical origin. The bacterial species studied shared important characteristics with respect to their FAME content: in all of them the saturated and unsaturated fatty acids of 16 carbon atoms were the predominant fatty acids. However, distinguishing features could be detected for each pathogen. Using either single linkage or complete linkage algorithms, strains were divided into four phena that corresponded to the different species, but showed a high degree of correlation among them. Although single linkage discriminated strains better within each phenum, complete linkage was more useful to establish the relationships among clusters. The results obtained support the idea that "Pasteurella" piscicida is related to members of the genera Vibrio and Aeromonas and indicate the need for exhaustive genetic studies to clarify the taxonomic position of this fish pathogen.
Animals↗
Capsular polysaccharide expressed by Pasteurella piscicida grown in vitro.
Pasteurella piscicida grown in a glucose-rich medium produces a capsule that can be see under light and electron microscopy. The capsular polysaccharide was purified and characterized by chemical and HPLC analysis. The polymer has the composition glucose/mannose/N-acetylgalactosamine/galacturonic acid/acetic acid in the molar ratios of approximately 2.5:1.3:0.5:0.4:2.5. The polysaccharide was immunogenic in rabbits and did not cross-react with antibodies against the O-antigen lipopolysaccharide.
Animals↗
Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment.
The stability of Pasteurella piscicida strains in seawater and sediment microcosms at different temperatures (6 and 20 degrees C) was investigated during a 1-month period. Three strains of P. piscicida showed similar survival kinetics. By a standard plate count method they survived in water and sediment for only 6 to 12 days, depending on the strain and type of microcosm. During this starvation period, the metabolic activity of the cells was reduced by more than 80%. Culturable cells of each P. piscicida strain persisted better in sediment than in water, as well as at 20 degrees C compared to 6 degrees C. However, in all the microcosms, the acridine orange direct counts remained at about 10(5) cells per ml during the experimental period, which demonstrated that P. piscicida possesses a capacity to enter a viable but not culturable state. Moreover, dormant cells were always resuscitated by the addition of fresh medium to the microcosms, since we recovered numbers of culturable cells similar to the acridine orange direct counts. These resuscitated cells exhibited the same respiration rate as that seen prior to the start of the experiments. Although the biochemical, physiological, and serological characteristics; lipopolysaccharides; membrane proteins; and plasmid content of P. piscicida strains were unaffected during the starvation conditions, the dormant cells were smaller (dwarf cells) and had increased surface hydrophobicity. The starved cells maintained their infectivity and pathogenic potential for fish, with 50% lethal doses similar to those of the original strains.
Animals↗
Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.
We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗