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Strains of Vibrio vulnificus, a marine bacterial species pathogenic for humans and eels, are divided into three biotypes, and those virulent for eels are classified as biotype 2. All biotype 2 strains possess one or more plasmids, which have been shown to harbor the biotype 2-specific DNA sequences. In this study we determined the DNA sequences of three biotype 2 plasmids: pR99 (68.4 kbp) in strain CECT4999 and pC4602-1 (56.6 kb) and pC4602-2 (66.9 kb) in strain CECT4602. Plasmid pC4602-2 showed 92% sequence identity with pR99. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1, which was self-transmissible, and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition.

Protein contact with the skin can be associated with 2 major clinical conditions: contact urticaria and protein contact dermatitis. This article reviews the pathogenesis, clinical pictures and the literature on the proteins having caused immunological occupation-related skin problems, i.e. fruits, vegetables, spices, plants, and woods; animal proteins; grains and enzymes, all affecting a wide variety of jobs. This is illustrated with some cases observed in the contact-allergy unit in Leuven.

The virulence for eels of Vibrio vulnificus biotype 2 serovar E (VSE) is conferred by a plasmid that codifies ability to survive in eel serum and cause septicaemia. To find out whether the plasmid and the selected chromosomal gene vvp plays a role in the initial steps of infection, the VSE strain CECT4999, the cured strain CT218 and the Vvp-deficient mutant CT201 (obtained in this work by allelic exchange) were used in colonization and virulence experiments. The eel avirulent biotype 1 (BT1) strain YJ016, whose genome has been sequenced, was used for comparative purposes. The global results demonstrate that the plasmid does not play a significant role in surface colonization because (i) CECT4999 and CT218 were equally chemoattracted towards and adherent to eel mucus and gills, and (ii) CT218 persisted in gills from bath-infected eels 2 weeks post infection. In contrast, mutation in vvp gene reduced significantly chemoattraction and attachment to eel mucus and gills, as well as virulence degree by immersion challenge. Co-infection experiments by bath with CECT4999 and CT201 confirmed that Vvp was involved in eel colonization and persistence in gills, because CECT4999 was recovered at higher numbers compared with CT201 from both internal organs of moribund fish (ratio 4:1) and gills from survivors (ratio 50:1). Interestingly, YJ016 also showed chemoattraction and attachment to mucus, and complementation of CT201 with BT1-vvp gene restored both activities together with virulence degree by immersion challenge. Additional experiments with algae mucus and purified mucin gave similar results. In conclusion, the protease Vvp of V. vulnificus seems to play an essential role in colonization of mucosal surfaces present in aquatic environments. Among the V. vulnificus strains colonizing fish mucus, only those harbouring the plasmid could survive in blood and cause septicaemia.

Vibrio vulnificus biotype 2 serovar E (VSE) is a bacterial pathogen that produces a haemorrhagic septicaemia called vibriosis in eels. Its ability to grow in blood is conferred by a recently described virulence plasmid [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.]. In this study, we analyzed the role of this plasmid together with the role played by the metalloprotease (Vvp) in the interaction between bacteria and eel innate immunity. To this end, we compared and statistically analyzed the differences in resistance to serum and mucus factors (complement, selected antimicrobial peptides, transferrin and lysozyme) and also to phagocytosis/opsonophagocytosis between one VSE strain and its derivatives: a plasmid-cured strain and a vvp-deficient mutant. The wild-type and the metalloprotease-deficient strains were resistant to both the bactericidal action of fresh serum and the phagocytosis and opsonophagocytosis by eel phagocytes, confirming that Vvp is not involved in resistance to eel innate immunity. In contrast, the cured strain was sensitive to both the bactericidal action of eel serum activated by the alternative pathway and phagocytosis/opsonophagocytosis. Since no plasmid-encoded ORF, with homology to known genes, is related to the resistance to innate immunity [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.], this function could be codified by one or more new genes. Further studies are underway to characterize the plasmid-encoded system responsible for V. vulnificus resistance to the innate immune system of eels.

Vibrio vulnificus is a bacterial species that is virulent for humans and fish. Human isolates are classified into biotypes 1 and 3 (BT1 and BT3) and fish isolates into biotype 2 (BT2). However, a few human infections caused by BT2 isolates have been reported worldwide (zoonosis). These BT2 human isolates belong to serovar E (SerE), which is also present in diseased fish. The aim of the present work was to characterize a new BT2 serovar [serovar A (SerA)], which emerged in the European fish-farming industry in 2000, by means of phenotypic, serological and genetic [plasmid profiling, ribotyping and random amplified polymorphic DNA (RAPD)] methodologies. The results confirmed that SerA constitutes a homogeneous O-serogroup within the species that shares plasmidic information with SerE. Like SerE, this new serogroup was resistant to fresh fish serum, as well as being highly virulent for fish. In contrast, it was sensitive to human serum and avirulent for mice, even after pretreatment with iron. The two serovars presented different biochemical profiles as well as specific patterns by ribotyping and RAPD analysis. In conclusion, SerA seems to constitute a different clonal group that has recently emerged within the species V. vulnificus, with pathogenic potential for fish but not for humans.

Vibrio vulnificus biotype 3 has been implicated as the causative pathogen of an ongoing disease outbreak that erupted in Israel in 1996. Recent work based on multi-locus sequence typing (MLST) showed that V. vulnificus biotype 3 is genetically homogeneous. The aim of this study was to investigate the existence of subpopulations within this homogeneous biotype by characterizing the surface antigens and analysing the sequence diversity of selected outer-membrane protein (OMP)-encoding genes. Rabbit antisera were prepared against biotype 1, 2 and 3 strains. The results of the slide-agglutination test, dot-blot assay (using fresh and boiled cells), and immunoblotting of lipopolysaccharides (LPS) and OMPs were evaluated. By slide-agglutination and dot-blot assays all biotype 3 strains agglutinated with the selected biotype 3 strain. This homogeneity was supported by immunoblot analysis of the LPS. Analysis of OMP patterns revealed that all three biotypes share a considerable number of common bands that are antigenically related. Cluster analysis of DNA sequence data from selected OMP-encoding genes showed that biotype 3 strains form a genetically distinct and homogeneous clone. The homogeneity of surface antigens and the lack of any sequence diversity among both housekeeping and OMP-encoding genes reaffirms the highly clonal nature of biotype 3 and suggests that it has only recently descended from the parent population of V. vulnificus.

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.

Yersinia ruckeri, the causal agent of enteric redmouth (ERM) disease, was isolated from epizootics that occurred in different Spanish rainbow trout, Oncorhynchus mykiss (Walbaum), farms in which vaccination against ERM had been performed. In all episodes, the most pronounced clinical signs exhibited by affected fish were severe haemorrhages in the mouth, eyes and around the vent. The isolates were identified as Y. ruckeri serovar I by 16S rRNA sequencing together with serological tests. They lacked motility and lipase activity and thus belonged to biotype 2, and were highly virulent for juvenile rainbow trout, both by intraperitoneal injection (from 3.1 x 10(2) to 6.3 x 10(3) cfu per fish) and bath challenge (5.1-7.3 x 10(6) cfu mL(-1)). This is the first description of Y. ruckeri serovar I biotype 2 causing disease in cultured trout in Spain vaccinated with commercial ERM vaccines. The occurrence of this emergent pathogen in Spanish continental aquaculture from its first isolation in 2001 to date is also documented.

The spread of the emerging pathogen Vibrio vulnificus biotype 2 serovar A in Danish anguilliculture is reported. Serovar A was originally isolated in a Spanish eel farm in 2000 and occurred in Denmark in the summer of 2004, affecting eels of 5-10 g body weight cultured in fresh water. The Danish eels showed clinical signs different from those reported for Spanish eels, such as severe haemorrhages in the head and gill region with necrosis of the soft tissues. Danish isolates were biochemically and serologically identical to Spanish serovar A strains and also highly virulent for eels by both intraperitoneal injection and immersion challenges. Vaccination with Vulnivaccine, a vaccine against V. vulnificus serovar E, cross-protected eels against serovar A. The LD(50) for experimentally infected vaccinated animals was significantly higher than for non-vaccinated animals.

Vibrio vulnificus can be divided into three biotypes, and only biotype 2, which is further divided into serovars, contains eel-virulent strains. We compared the genomic DNA of a biotype 2 serovar E isolate (tester) with the genomic DNAs of three biotype 1 strains by suppression subtractive hybridization and then tested the distribution of the tester-specific DNA sequences in a wide collection of bacterial strains. In this way we identified three plasmid-borne DNA sequences that were specific for biotype 2 strains irrespective of the serovar and three chromosomal DNA sequences that were specific for serovar E biotype 2 strains. These sequences have potential for use in the diagnosis of eel vibriosis caused by V. vulnificus and in the detection of biotype 2 serovar E strains.

The nucleoside 2-amino-9-(2-deoxy-beta-d-ribofuranosyl)-7,8-dihydro-8-oxo-purine (dJ) was obtained in eight steps from 2'-deoxyguanosine. The appropriate protected phosphoramidite was synthesized and incorporated into DNA oligonucleotides. The thermal stability of heteroduplexes containing 2-amino-8-oxopurine (J) was investigated by UV-thermal denaturation experiments. The results obtained can be interpreted by the base pairing schemes involving the two edges of dJ depending on the anti and syn orientations.

Vibrio vulnificus can be divided into two groups on the basis of pathogenesis. Group 1 is pathogenic only to humans, whereas group 2 is pathogenic to eels and occasionally to humans. Although both groups produce a 50-kDa cytotoxin-hemolysin (V. vulnificus hemolysin; VVH), the toxins are different. In the present study, the nucleotide sequence of the toxin gene (vvhA ) of strain CDC B3547 (a group 2 strain) was determined, and the deduced amino acid sequence was compared to that of strain L-180 (a group 1 strain). The nucleotide sequence of vvhA of strain CDC B3547 was about 96% identical with that of strain L-180, which results in a difference of 3 amino acid residues in the C-terminal lectin domain of VVH. Nevertheless, two primer sets for polymerase chain reaction could be designed to differentiate the toxin gene of each strain. When 27 V. vulnificus clinical isolates were tested, group 1 strains (9 strains) were shown to react only to the primers designed for vvhA of strain L-180; whereas, the gene of group 2 strains (18 strains) could be amplified with the primers for vvhA of strain CDC B3547. These findings may lead to development of a novel genetic grouping system related to the virulence potential or to the host range.

The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V. vulnificus groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control. V. vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.

The immunogenic antigens of Vibrio vulnificus serovar E were investigated in the eel. Fish were vaccinated by immersion with Vulnivaccine (V), revaccinated 2 years later by intraperitoneal injection (RV) and bath infected 15 days post-revaccination (RVI). The specific immune response in serum was followed in all groups, and selected sera were used for immunostaining of surface (SA) and extracellular antigens (ECA). Bacteria were grown in iron-rich (TSB and MSWYE) and iron-poor media (TSB and MSWYE plus human transferrin (TSB-T and MSWYE-T)) as well as eel serum (ES), and their SA and ECA were extracted and electrophoretically analysed. Cells grown in MSWYE-T and ES presented the same antigenic profiles, which suggests that iron-restriction is the main growth-limiting factor in vivo. The electrophoretic pattern of SA, but not that of ECA, varied with iron-availability in the growth medium. Further, SA extracted from bacteria grown in iron restriction were strongly immunogenic for eels, especially after vaccination and infection. Among the immunogenic antigens over expressed in iron-restriction, three outer membrane proteins of around 70-80 kDa, including the putative receptor for vulnibactin, together with the rapid and slow migrating forms of the lipopolysaccharide (LPS), were identified. The response was not so evident in the case of capsule, which was not clearly stained with any of the eel sera. With respect to ECA, two proteins, identified as the V. vulnificus protease (Vvp) and the major outer membrane protein (OMP), probably liberated to the medium after cell death, were recognised by RV and, more strongly, by RVI sera. The specific antibodies against the mentioned OMPs, LPS bands and the Vvp may contribute to the protection of vaccinated eels against infection, giving a reasonable explanation for the high effectiveness of Vulnivaccine.

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.

Vulnivaccine, a vaccine against vibriosis caused by Vibrio vulnificus serovar E (formerly biotype 2), confers acceptable levels of protection to eels after its administration by prolonged immersion in three doses. Recently, a new pathogenic serovar, named serovar A, has been isolated from vaccinated eels in a Spanish freshwater eel farm. The main objective of this work was to design a bivalent vaccine, and to study its effectiveness against the two pathogenic serovars. With this aim, eels weighing around 20 g were immunised with the bivalent vaccine by oral and anal intubation, intraperitoneal injection (i.p.) and prolonged immersion. The overall results indicated that: (i) the new vaccine delivered by oral and anal intubation induced protection levels higher than 80%, to that achieved after i.p. vaccination; (ii) oral and anal vaccination induced a significant systemic and mucosal immune response; (iii) the protection after vaccination by whichever routes was related to antibody titres in plasma; (iv) mucosal and systemic compartments showed different kinetics of antibody production; (v) evidence for passive transfer of antibodies from plasma to gut mucus were found after i.p. and anal vaccination, and finally, (vi) vaccination did not enhance the production of lysozyme, in plasma or mucus. In conclusion, this new vaccine is effective in protecting eels against vibriosis caused by the two eel-pathogenic serovars of V. vulnificus, the oral delivery system is a promising way which may be used in intensive culture facilities during the whole growth period of eels.

Vibrio vulnificus serovar E, a bacterial pathogen for eels cultured in intensive systems, is transmitted through water and enters into new hosts mainly via gills. The main objective of this work was to study the kinetics of antibody production to V. vulnificus in serum and mucus and their relationship with protection after vaccination. To quantify local mucus antibodies, a new "in situ" dot blot immunoassay using image analysis has been developed. This assay was applied to measure antibody production in the skin zone next to the gills. We found that (i) the immune response in mucus was faster (peak at days 3-4) and shorter in duration (titres significantly elevated up to day 5 and 11 for skin zone next to the gills and for general cutaneous mucus, respectively) than in serum (peak at day 7; titres significantly elevated for more than 25 days); (ii) the exposure of vaccinated eels with basal levels of local antibodies to sub-lethal dose of the pathogen stimulated a more lasting secreted antibody production (for more than 14 days); (iii) protection and antibody levels in serum were clearly correlated, and (iv) immunised eels with basal levels of serum antibodies and maximal levels of local antibodies were partially protected.

Vibrio vulnificus serovar E (formerly biotype 2) is the etiologic agent that is responsible for the main infectious disease affecting farmed eels. Although the pathogen can theoretically use water as a vehicle for disease transmission, it has not been isolated from tank water during epizootics to date. In this work, the mode of transmission of the disease to healthy eels, the portals of entry of the pathogen into fish, and their putative reservoirs have been investigated by means of laboratory and field experiments. Results of the experiments of direct and indirect host-to-host transmission, patch contact challenges, and oral-anal intubations suggest that water is the prime vehicle for disease transmission and that gills are the main portals of entry into the eel body. The pathogen mixed with food can also come into the fish through the gastrointestinal tract and develop the disease. These conclusions were supported by field data obtained during a natural outbreak in which we were able to isolate this microorganism from tank water for the first time. The examination of some survivors from experimental infections by indirect immunofluorescence and scanning electron microscopy showed that V. vulnificus serovar E formed a biofilm-like structure on the eel skin surface. In vitro assays demonstrated that the ability of the pathogen to colonize both hydrophilic and hydrophobic surfaces was inhibited by glucose. The capacity to form biofilms on eel surface could constitute a strategy for surviving between epizootics or outbreaks, and coated survivors could act as reservoirs for the disease.

The field results of a vaccination programme against Vibrio vulnificus serovar E (biotype 2) in a Spanish eel farm are reported. A total of 9.5 million glass eels were vaccinated from January 1998 to March 2000 by prolonged immersion followed by 2 subsequent reimmunisations after 12 to 14 and 24 to 28 d, respectively. The acquired protection and the immune response against serovar E were estimated over a period of 6 mo after vaccination. A similar vaccination schedule was conducted with elvers in a Danish eel farm. In this case, the acquired protection and the immune response against serovar E and the new eel-pathogenic serovars, recently described in Denmark, were evaluated over a short term. The overall results show that the vaccine against V. vulnificus serovar E induces a satisfactory protective immunity during the main growth period of eels (around 6 mo) with a relative percentage survival of 62 to 86% and protects them against the new eel-pathogenic serovars. Vaccination of eels by immersion seems to be the best strategy to prevent diseases caused by V. vulnificus.

Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids--inactivated extracellular products--from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide [LPS]) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion). To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activity in mucus and serum. To evaluate protection, we calculated the relative percent survival (RPS) after intraperitoneal (i.p.) injection and bath challenge of the pathogen. The overall results indicate that: (1) capsular antigens seem to be essential for protective immunization; (2) vaccines confer the highest protection when administered by i.p. injection; (3) booster is needed to achieve good protection by immersion; (4) enriching the vaccine with toxoids enhances protection to optimal levels (RPS values around 70 to 100%, depending on the delivery route); and (5) the protective effect in serum and mucus depends on the route of administration and seems to be related to the production of specific antibodies.

The applications of an indirect fluorescent antibody technique (IFAT), developed to detect and enumerate the pathogenic bacterium Vibrio vulnificus serovar E from water and clinical samples, are described. This technique proved accurate for detecting V. vulnificus, even under starvation conditions and in the non-culturable state, and could differentiate this species from other bacteria which share the same habitats. The IFAT was successfully used to diagnose vibriosis from naturally- and artificially-infected eels. The overall data suggest that applying this technique properly in environmental and epidemiological/epizootiological studies could significantly increase our knowledge of this bacterium.

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.

In this paper, the ferric-reductase activities of Vibrio vulnificus were investigated. This species comprises two biotypes pathogenic for humans and eels that are able to express different mechanisms for iron acquisition. All strains of both biotypes used in this study were able to reduce ferric citrate, irrespective of the iron levels in the growth medium. Some variation in the degree of reduction was observed among the strains, with the highest values corresponding to one acapsulated environmental strain of biotype 1. When cell fractions were tested, only those from periplasm and cytoplasm showed reductase activity whereas no activity was detected in membranes. Low temperatures inhibited these activities in both whole cells and cell fractions. At least six bands with ferric-reductase activity were identified in all strains using native polyacrylamide gels. These data demonstrate that the two biotypes of V. vulnificus produce similar ferric-reductases mainly located in the periplasm and cytoplasm and these could be involved in iron acquisition.

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.

Vibrio vulnificus biotype 2 (serovar E) is a primary eel pathogen. In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium. We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30 degrees C). Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25 degrees C for microcosms at 0.3 and 0.5% and 12 degrees C for microcosms at 1.5 to 3.8%). In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis. Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years. The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones. Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice. Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced. In conclusion, V. vulnificus biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water. Moreover, the aquatic ecosystem is one of its reservoirs.

In Vibrio vulnificus, virulence for eels is associated with serovar E strains. In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains. Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.

High mortality among laboratory cultured Iberian toothcarp Aphanius iberus occurred in February 1997 in Valencia (Spain). The main signs of the disease were external haemorrhage and tail rot. Bacteria isolated from internal organs of infected fish were biochemically homogeneous and identified as Vibrio parahaemolyticus. The bacteria were haemolytic against erythrocytes from eel Anguilla anguilla, amberjack Seriola dumerili, toothcarp A. iberus and humans, and were Kanagawa-phenomenon-negative. Infectivity tests showed that the virulence for A. iberus was dependent on salinity. Finally, all strains were virulent for amberjack and eel.

In this work, the high affinity iron-acquisition systems displayed by virulent and avirulent strains of Vibrio damsela have been investigated. This species is an autochthonous member of marine ecosystems that can behave as an opportunistic pathogen for fish and mammals. All strains tested (i) were able to grow under the restricted conditions imposed by the iron chelators transferrin (Tf) and EDDHA, (ii) secreted siderophores of hydroxamic type, other than aerobactin and desferal, that were able to stimulate the growth of the auxotroph mutant Arthrobacter flavescens JG9, and (iii) expressed common iron-regulated outer membrane proteins (IROMPs). No change in LPS patterns was observed in response to iron restriction. Results from the assays with transferrin suggest that these siderophores could be utilized to sequester iron from Tf, a protein for which no surface receptor was detected in any strain. In summary, the overall data demonstrate that V. damsela expresses siderophore-mediated iron-uptake systems. These systems are probably involved in the survival of the species in the different environments that it can colonize, i.e. water and several vertebrate hosts.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.

The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs] and outer membrane proteins [OMPs]) was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB+ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB- strains antigenically diverse that either exhibited a heterogeneous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB+ strains, whereas major OMPs detected in PAB- strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.

The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European eels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strains. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus. Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus.

Lipopolysaccharides (LPS) of 11 strains of Vibrio vulnificus biotypes 1 and 2, isolated from an eel farm, and of 10 reference strains, were examined by SDS-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. LPS samples were obtained from whole-cell lysates, outer membrane fragments, and extracellular products. By silver staining, only a diffuse band of low-molecular weight could be visualized in all cases except for a biotype 1 strain isolated from water. However, immunoblotting with antisera obtained against strains of biotypes 1 and 2 from eels allowed visualization of multiple O-polysaccharide chains. All biotype 2 strains, independently of their origins, belonged to the same serotype and presented the same LPS profile, whereas eel isolates of biotype 1 were serologically identical and different from the rest of tested strains of biotype 1. This is the first report of LPSs with a ladder-like structure in Vibrio vulnificus.

Iron uptake mechanisms were investigated in different species of Salmonella isolated from environmental waters. All strains examined were able to grow in the presence of high concentrations (10 mM) of the iron chelator EDDA. All strains excreted phenolate and hydroxamate siderophores, as assessed by bioassays and chemical tests. Bioassays with different indicator strains showed that all Salmonella strains can cross-feed other Enterobacteria, as well as mutants of Salmonella typhimurium deficient in the Enterobactin system, suggesting that this siderophore may be produced by the environmental Salmonella strains. The siderophore aerobactin may also be produced by one of the strains, according to the bioassays results. The same pattern of outer membrane proteins are synthesized under iron-limiting conditions in all species tested, which suggests a similarity of iron uptake systems in many species of Salmonella. This system could be also of great importance in the survival of these bacteria in natural waters, as well as in possible pathogenic mechanisms.

The cell envelope proteins of 89 environmental Vibrio cholerae non O1 strains isolated from lake and coastal waters near Valencia, Spain, and six Vibrio cholerae strains from culture collections were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Considerable heterogeneity was found in the major proteins of the environmental non-O1 strains, but bands between 25,000 and 48,000 daltons were observed in the majority of the strains. Estimated relative mobilities of the total protein profile ranged between 11 and more than 100 Kd. Cluster analysis revealed four groups of strains distinguishable by presence or absence of high and low molecular weight proteins. After treatment with Sarkosyl, the outer membrane proteins (OMPs) were characterized in all strains by densitometric methods. They ranged from 19 to 87 Kilodaltons, and corresponded to the major proteins observed in the total membrane preparations. The major OMP most frequently found had a molecular weight around 37 Kd, similar to that of porins in other Gram-negative bacteria. The OMP composition varied in response to culture medium and growth phase. Generally the OMP expression was affected only in a quantitative way by the growth phase while the growth medium had both a qualitative and a quantitative effect.

A taxonomic study by means of DNA homology and the most recent phenetic criteria has been carried out in 11 environmental strains of Vibrio parahaemolyticus and 4 of Vibrio pelagius, previously ascribed to the respective phenons in a numerical taxonomy study. The strains of V. parahaemolyticus showed genetic homogeneity, a guanine plus cytosine content (G + C) of 46% and 61% homology with V. parahaemolyticus type strain ATTC 17802. They exhibited phenetic atypicalities but could be ascribed to the species V. parahaemolyticus. The strains of V. pelagius behaved similarly in regard to the phenotype, but were genetically heterogeneous; they showed a % G + C of 44-46 and were assigned to V. splendidus-V. pelagius homology group.

Most probable numbers of zooplankton-associated Vibrio spp. and V. cholerae were determined in Albufera lake, Spain, and in its coastal receiving waters throughout a year. Highest counts of 10(5) bacterial cells/g of plankton were associated to high temperatures and were also related to the kind of water. All isolates were non-01 serovars, and most belonged to Heiberg groups I and II.