A challenge model for Tenacibaculum maritimum infection in turbot, Scophthalmus maximus (L.).
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Animals↗
Biomedical subjects
A E Toranzo
Publications and source records attributed to A E Toranzo.
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↗
Lactococcus garvieae in wild Red Sea wrasse Coris aygula (Labridae).
Lactococcus garvieae infection in wild wrasse Coris aygula is reported, and the serological and molecular characteristics of the isolate are described. This is the first evidence of the presence of this pathogen in the Red Sea, and it follows the recent diagnosis of Mycobacterium marinum and Streptococcus iniae in wild fish from the same region. Whether all 3 pathogens are strains endemic to the Red Sea, or recent introductions into the region, remains to be determined, but their appearance over a period of a few years in wild fish populations in the northern Red Sea is consistent with an emerging trend affecting marine organisms on a global level in areas subjected to intense anthropogenic impacts.
Animals↗
Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot, Scophthalmus maximus (L.), larvae.
A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.
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↗
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.
Animals↗
Presence of phospholipase-D (dly) gene coding for damselysin production is not a pre-requisite for pathogenicity in Photobacterium damselae subsp. damselae.
The presence of the phospholipase-D (dly) gene as pre-requisite for virulence of Photobacterium damselae subsp. damselae for poikilotherm and homoiotherm animals was investigated in a total of 17 strains isolated from fish, shellfish, mammals and seawater. With this aim, we developed two PCR protocols. A simple PCR using primers flanking the almost complete dly gene, and a multiplex-PCR using two sets of primers directed to internal fragments of the dly and 16S rRNA genes. Only six of the 17 Ph. damselae subsp. damselae strains studied harboured the dly gene regardless of their haemolytic activity against sheep or rabbit erythrocytes as well as their virulence for mammals and marine fish. In fact, all strains but one were pathogenic for one or both animals, with LD(50)values ranging from 1x10(3)and 3x10(5)bacteria for turbot, and 2x10(6)and 8x10(7)cells for mice. The PCR results were corroborated in dot blot hybridization experiments employing a DNA probe directed to an internal region of the dly gene. From the data obtained in this work, we can conclude that the presence of the dly gene is not an indicative of the pathogenicity of Ph. damselae subsp. damselae and, therefore, the role of damselysin as the main virulence factor of this marine bacterium for poikilotherm and homoiotherm hosts should be re-evaluated.
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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↗
Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae.
The transmission through water of the disease caused by the fish pathogen, Photobacterium damselae subsp. damselae, as well as the role of the skin mucus in the initial steps of the infection, have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those virulent by the intraperitoneal route were infective through water. Moribund fishes showed the typical signs of the disease: haemorrhaged areas on the body surface and ulcerative lesions with mucus degradation. These results suggest that the pathogen can be transmitted to fish through water and use the skin as a portal of entry.
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.
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16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.
The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.
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Survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater under starvation conditions.
The survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater microcosms, with and without sediment, was investigated. The strains survived as culturable bacteria at 14 and 22 degrees C for at least 1 year, and infectivity for fish was maintained. At 5 degrees C, cells lost culturability on solid media, but this was recovered when the temperature was increased to 22 degrees C. Finally, morphological changes in the bacterium (rod to coccus), and production of vesicles and extracellular material were observed during the time of starvation. The overall results suggest that seawater and sediment can act as reservoirs for these virulent strains.
Animals↗
Edwardsiellosis in wild striped bass from the Chesapeake Bay.
The first epizootic of edwardsiellosis, caused by Edwardsiella tarda, is described. The epizootic occurred in the Chesapeake Bay, Maryland (USA) during the summer and autumn of 1994, and affected wild adult striped bass (Morone saxatilis). Clinical signs included numerous irregular coalescing hemorrhagic ulcers on the body and fins that were distinctly malodorous. Internally, the body cavity was filled with abundant yellowish or sanguinous mucoid fluid, and the visceral organs had multiple tiny white foci. The intestines contained thick white opaque mucus. Histopathological lesions included ulcerative dermatitis, cardiac endothelial hyperplasia, and necrotic foci and granulomata in multiple organs. A bacterium isolated in pure culture was characterized taxonomically and serologically as the wild-type or classical biotype of E. tarda: In infectivity trials, it was pathogenic for striped bass, gilthead seabream (Sparus aurata), and turbot (Scophthalmus maximus) with an LD50 of about 10(5) cells; however, the isolate was non-virulent for mice (LD50 > 10(8) cells). The isolate also was resistant to the bacteriolytic activity of normal fish skin mucus.
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Immunization with bacterial antigens: Vibrio infections.
Within the genus Vibrio, the species causing the most economically important diseases in marine culture are Vibrio anguillarum, V. ordalii, V. salmonicida and V. vulnificus biotype 2. For these bacterial fish pathogens host range, clinical importance, virulence mechanisms, the antigenic variants relevant to vaccination, the existence of genetic intraspecific diversity and the available vaccines including commercial or domestically produced will be described. Among the 10 serotypes described in V. anguillarum, only serotypes O1, O2 and O3 have been associated with mortality in a great variety of farmed and feral fish worldwide. Whereas serotype O1 is a very homogeneous group from the biochemical, serological and genetic stand-point, within serotype O2 and O3 two antigenic entities have been detected. Moreover these two serotypes present a remarkable genetic heterogeneity. However, many of the available commercial vibriosis vaccines include in their formulations only V. anguillarum serotype O1 in combination with V. ordalii (formerly V. anguillarum biotype 2). In addition no commercial vaccine provides information about the subgroup(s) used as representative of V. anguillarum O2. Recently, Vibrio species taxonomically related to V. anguillarum (VAR) have been isolated from diseased fishes. An extensive characterization of these VAR organisms allowed us to distribute them into at least seven O-serogroups. The inclusion of representative VAR strains in the vibriosis vaccines need to be discussed. V. ordalii, V. salmonicida and V. vulnificus are homogeneous species with respect to biochemical reactions, serology and degree of virulence, possess a narrow host range and seem to be restricted to some geographic areas. Although iron acquisition systems can be involved in the virulence mechanisms of these pathogens, only in V. anguillarum has it been clearly demonstrated that the ability to scavenge iron from the host is a crucial virulence determinant. The role of exotoxins and cell surface associated properties in the Vibrio infections remains to be elucidated.
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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.
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