Transmission to eels, portals of entry, and putative reservoirs of Vibrio vulnificus serovar E (biotype 2)

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.     

Protocol for Specific Isolation of Virulent Strains of Vibrio vulnificus Serovar E (Biotype 2) from Environmental Samples

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.     

Heterogeneity among Isolates of Vibrio vulnificus Recovered from Eels (Anguilla anguilla) in Denmark

The findings of this study demonstrate that Vibrio vulnificus isolates recovered from diseased eels in Denmark are heterogeneous as shown by O serovars, capsule types, ribotyping, phage typing, and plasmid profiling. The study includes 85 V. vulnificus isolates isolated from the gills, intestinal contents, mucus, spleen, and kidneys of eels during five disease outbreaks on two Danish eel farms from 1995 to 1997, along with a collection of 12 V. vulnificus reference strains. The results showed that more than one serovar may be capable of causing disease in eels and that these isolates are genetically heterogenous as shown by ribotyping. Ribotyping also showed that the same isolates may persist in an eel farm and cause recurrent outbreaks. Phage typing did not correlate with ribotyping or serotyping. However, we observed that 26 of 28 isolates, which were not susceptible to any of the phages, showed the same ribotype, O serovar, and capsule type. This suggests that these isolates may possess features that make them resistant to lysis by the phages used in this study. Ninety-three of 97 isolates harbored between one and three high-molecular-weight plasmids which previously had been suggested to be associated with eel virulence. The subdivision of V. vulnificus into two biotypes based on the indole reaction can no longer be supported, since 82 of 97 isolates in this study were indole positive, and a subdivision into serovars appears to be more correct.     

Protocol for specific isolation of virulent strains of Vibrio vulnificus serovar E (biotype 2) from environmental samples

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.     

Multiplex PCR Assay for Detection of Vibrio vulnificus Biotype 2 and Simultaneous Discrimination of Serovar E Strains

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.     

Occurrence of Vibrio vulnificus Biotypes in Danish Marine Environments

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 10⁴ U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolated V. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificus in European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.     

Comparative Study of Biological Properties and Electrophoretic Characteristics of Lipopolysaccharide from Eel-Virulent and Eel-A virulent Vibrio vulnificus Strains

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.     

Novel host‐specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host‐specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron‐regulated gene overexpressed in eel blood during artificially induced vibriosis both in vitro and in vivo. Interestingly, homologues to vep20 were identified in the transferable plasmids of two fish pathogen species of broad‐host range, Vibrio harveyi (pVh1) and Photobacterium damselae subsp. damselae (pPHDD1). These data suggest that Vep20 belongs to a new family of plasmid‐encoded fish‐specific transferrin receptors, and the acquisition of these plasmids through horizontal gene transfer is likely positively selected in the fish‐farming environment. Moreover, we propose Ftbp (fish transferrin binding proteins) as a formal name for this family of proteins.     

Identification of DNA Sequences Specific for Vibrio vulnificus Biotype 2 Strains by Suppression Subtractive Hybridization

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.     

Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a zoonotic pathogen able to cause diseases in humans and fish that occasionally result in sepsis and death. Most reviews about this pathogen (including those related to its ecology) are clearly biased towards its role as a human pathogen, emphasizing its relationship with oysters as its main reservoir, the role of the known virulence factors as well as the clinic and the epidemiology of the human disease. This review tries to give to the reader a wider vision of the biology of this pathogen covering aspects related to its phylogeny and evolution and filling the gaps in our understanding of the general strategies that V. vulnificus uses to survive outside and inside its two main hosts, the human and the eel, and how its response to specific environmental parameters determines its survival, its death, or the triggering of an infectious process.     

Indole-positive Vibrio vulnificus isolated from disease outbreaks on a Danish eel farm

Vibrio vulnificus was isolated in 1996 from 2 disease outbreaks on a Danish eel farm which used brackish water. A characteristic clinical sign was extensive, deep muscle necrosis in the head region. V. vulnificus was isolated from kidney, mucus, spleen, gill and intestine of diseased eels. Thirty-two isolates were examined phenotypically and serologically for pathogenicity to eels and for correlation to ribotype and plasmid profile. Biochemically, the isolates showed properties similar to those described previously for eel-pathogenic strains of V. vulnificus, with the exception of indole production. Virulence was evaluated by LD50 (the 50% lethal dose), which ranged from < 9.4 x 10(3) to 2.3 x 10(5) CFU (colony-forming units) per fish. The isolates which were lethal for eels showed identical ribotypes and serotypes. A relationship between certain plasmids and virulence was not found. A serotyping system based on lipopolysaccharide (LPS)-associated O antigen type and on carbohydrate capsule antigens showed that the eel-virulent isolates shared a common LPS-based homogeneous O serogroup and a capsule antigen. V. vulnificus serovar O4 and capsule type 9 was identical serologically to the Japanese isolate ATCC 33149 and was the agent responsible for the disease outbreaks that occurred on the Danish eel farm. Despite absence of antibiotic resistance, treatment had little effect and disease reoccurred.     

Role of the metalloprotease Vvp and the virulence plasmid pR99 of Vibrio vulnificus serovar E in surface colonization and fish virulence

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.     

Relationships between locomotor behavior, morphometric characters and thyroid hormone levels give evidence of stage-dependent mechanisms in European eel upstream migration

In order to decipher movements during freshwater eel colonization, we experimentally characterized individual locomotor behavior of two eel life history stages: elvers and yellow eels. A ramp located at the flume tank upstream side required a specific locomotor behavior to be ascended. Placing individually tagged eels in the middle of the tank three times successively tested behavioral consistency. Eels climbing the ramp on each trial were classified as "upstream climbers" whereas eels settling in the tank middle were classified as "inactive". Both stages exhibited these two opposite consistent behaviors. However, elvers were predominantly "upstream climbers" (58.1%) whereas yellow eels were predominantly "inactive" (79.6%). We measured morphometric characters and thyroid hormones to determine if upstream activity was related to body condition and thyroid status. Elver upstream climbers had higher body condition as well as higher thyroxine (T(4)) and triiodothyronine (T(3)) levels compared with inactive elvers. Yellow eel upstream climbers had lower body length as well as higher T(3) and (T(3):T(4)) ratio compared with inactive yellow eels. This indicated that the physiological release factors for eel upstream migration may be stage dependent. For elvers, high thyroid gland activity, together with high body condition, may be the physiological release factors for migration. In contrast, for yellow eels, physiological stress may be the release factor with an increase in T(4) deiodination activity in the smallest eels. Our study revealed inter-stage and intra-stage locomotor behavior plasticity and suggested stage-dependent opposite impacts of physiological condition on eel upstream migration.     

Regulation systems of protease and hemolysin production in Vibrio vulnificus

Vibrio vulnificus, a gram‐negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema‐forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone‐like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.     

Follower fish of the goldspotted eel <Emphasis Type="Italic">Myrichthys ocellatus</Emphasis> with a review on anguilliform fish as nuclear species

In a nuclear-follower fish foraging association, the follower benefits from food uncovered or flushed out when the nuclear fish disturbs the bottom, while nuclear species generally do not seem to be benefiting. Among nuclear species, eels (anguilliform fish) are known to be one of the most represented groups. Here we investigated the frequency and time duration of foraging associations among the goldspotted eel Myrichthys ocellatus and reef fish in a subtropical marginal reef. In addition, we reviewed nuclear eel species and their followers described in the literature. From a total of 211 goldspotted eels observed, seven follower species were recorded in 19% of the samples. The average time of the following associations per species ranged from 40 to 190 s. Four species were reported for the first time as M. ocellatus followers (Bodianus rufus, B. pulchellus, Stephanolepis hispidus, and Serranus baldwini) and three of them have never been reported in the literature as eel followers (B. pulchellus, S. hispidus, and S. baldwini). The literature describes 13 eel species acting as nuclear for 66 fish species, represented mainly by groupers and sea basses. The size of the eel was not correlated with the size of its follower and neither with the number of described follower species. The nuclear role of eels is likely to be an important component of the trophic ecology of small and medium-sized macrocarnivore fish.     

Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla.

Herpesvirus of eel Herpesvirus anguillae (HVA) was isolated repeatedly from farmed eel of an outwardly healthy stock, but virus isolation was much greater in an experimental group of fish that were injected with dexamethasone. The results suggest that HVA can establish a latent infection in eel. Previous exposure of these eels to HVA virus was shown by detection of HVA-specific antibodies. These eels did not show clinical signs after a secondary infection with HVA. Tracing of seropositive eel stocks, which had previous contact with HVA, and of HVA carrier fish can be useful to control disease outbreaks due to HVA infection.     

Recruitment abundance estimation: Role of glass eel (Anguilla anguilla L.) response to light

Most fish populations are declining worldwide and their management would benefit from a better estimation of recruitment. In glass eels, field studies suggest that estuarine migratory glass eels are sensitive enough to light to change their vertical location according to factors such as water turbidity and/or moon brightness. The response of glass eel (Anguilla anguilla L.) to light was tested in the laboratory using boxes where fish could choose between a lit and an unlit side. Responses were quantified as the proportion of glass eels remaining in the unlit chamber. Decreasing light levels were used and tested on different “age” glass eels (“age” in days since capture). In addition, measures of light at different depths of the water column were carried out in the Adour estuary (43°30′ N, 1°30′ W). The glass eel light avoidance level was lower in non-pigmented glass eel (less than 10 ^− 10 W cm ^− 2), than in pigmented ones (10 ⁻⁹-10 ^− 8 W cm ^− 2). These results and field data on the measurement of light energy in the water column of Adour estuary are compared with previously published data on the estuarine migration of glass eel.     

Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan

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.     

Sediment and Vegetation as Reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.     

Escapement success of silver eels from a German river system is low compared to management‐based estimates

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