Vulnificus Serovar Research Articles

Microbial and histopathological study of the vibriosis caused by Vibrio vulnificus serovar E in eels: The metalloprotease Vvp is not an essential lesional factor

Vibrio vulnificus biotype 2 serovar E (Bt2-serE) is a zoonotic pathogen that causes a haemorrhagic septicaemia in eels, called warm water vibriosis. The main objective of the present work was to study the onset of the eel vibriosis from the microbiological and histopathological viewpoint, as well as to ascertain the role of the protease Vvp as a lesional factor by comparing the histopathological lesions caused by the wild strain and its vvp deficient derivative. The wild-type strain was observed to attach to the gills, where it multiplied following saturation dynamics, subsequently invading the blood stream and reaching the internal organs. Here it reached population sizes that are notably lower than those associated with other fish septicaemia. Parallel to bacterial growth, there was a notable decrease in haematocrit values and haemoglobin concentration in blood as well as extensive haemorrhages in all the analysed organs. The main histopathological lesions were detected in the head kidney in the form of extensive necrosis affecting the haematopoietic tissue. Very few bacteria were visualized in the different organs, most of which were close to blood cells and capillary vessels, which is compatible with the results obtained in the microbiological study. The same lesions were produced when extracellular products (ECPs) were injected instead of bacteria or when the vvp-defective mutant or its ECPs were injected. The overall results suggest that the pathology caused by V. vulnificus in the eel is not caused by massive bacterial growth in the blood and internal organs but, rather, by the effect of potent toxic factors other than the metalloprotease, which have yet to be determined.

Role of the virulence plasmid pR99 and the metalloprotease Vvp in resistance of Vibrio vulnificus serovar E to eel innate immunity

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.

Phenotypic and genotypic characterization of a new fish-virulent Vibrio vulnificus serovar that lacks potential to infect humans

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.

A method to diagnose the carrier state of Vibrio vulnificus serovar E in eels: Development and field studies

The pathogen Vibrio vulnificus serovar E (VSE) has been related to both human infections and to epizootics causing high mortality in brackish water eel farms. To control the spread of the eel vibriosis and prevent VSE transmission to humans we designed and tested a protocol to detect carriers, which involves isolating the pathogen. To identify the organs where VSE persists in survivors we infected eels with different degrees of immunity against the pathogen (non-immune [NI], immune [I, eels vaccinated 1 year before] and freshly vaccinated [V]) by bath challenge. Then, we followed the pathogen survival in selected external and internal organs for 72 h post-infection. VSE was isolated by using previously described protocols [Sanjuan, E., Amaro, C., 2004. Protocol for specific isolation of virulent strains of V. vulnificus serovar E (biotype 2) from environmental samples. Appl. Environ. Microbiol. 70, 7024–7032.] and identified by a new specific PCR methodology [Lee C.-T., Amaro, C., Sanjuan, E., Hor, L-I., 2005. Identification of DNA sequences specific for V. vulnificus biotype 2 strains by suppression subtractive hybridization. Appl. Environ. Microbiol. 71: 5593–5597.]. The pathogen survival depended on the immunological status of the animal. Thus, VSE persisted in the gills and spleen of NI and I eels but was eliminated by the immune system of V eels in less than 9 h post-infection. We selected gills as the organ to be sampled and designed a whole protocol for carrier detection. The feasibility of the protocol was demonstrated in field studies after the positive isolation of the pathogen from asymptomatic carriers without killing the animals.

Vibrio vulnificus serovar A: an emerging pathogen in European anguilliculture

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.

Vaccination of market-size eels against vibriosis due to Vibrio vulnificus serovar E

Vaccination with Vulnivaccine at eel farms has been previously shown to protect cultured eels against vibriosis caused by Vibrio vulnificus serovar E for more than 1 year. The reported protocol included an initial vaccination by triple prolonged immersion at the glass-eel stage together with one optional oral booster at the elver stage. However, eels at the market-size stage (around 150 g body weight) can suffer stress-related vibriosis after handling and transport to the selling facilities, which implies a serious risk for consumer health. The main objective of this work was therefore to develop an effective re-vaccination procedure, useful for preventing stress-related vibriosis and zoonosis in market-size eels. To this end, eels, primed 2 years before, were reimmunised by prolonged immersion with a very low dose of Vulnivaccine, a procedure commercially applicable to large fish. The protection, the immune response in plasma, skin mucus and bile as well as the bactericidal activity of the collected body fluids were evaluated in reimmunised and control animals (non-reimmunised) during a 30-day period. Reimmunisation significantly increased antibody titres in plasma, mucus and bile, as well as the bactericidal activity of these fluids, and reduced the mortality rate after bath challenge with the pathogen. In conclusion, the prolonged immersion in a very high dilution of Vulnivaccine can protect market-size eels against stress-related vibriosis and, consequently, significantly reduce additional risks of zoonosis.

Immunogenic antigens of the eel pathogen Vibrio vulnificus 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.

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

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.

Efficacy of a bivalent vaccine against eel diseases caused by Vibrio vulnificus after its administration by four different routes

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.

The kinetics of antibody production in mucus and serum of European eel ( Anguilla anguilla L.) after vaccination against Vibrio vulnificus: development of a new method for antibody quantification in skin mucus

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.

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.

Field testing of a vaccine against eel diseases caused by Vibrio vulnificus.

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.

A highly homologous 68 kbp plasmid found in Vibrio vulnificus strains virulent for eels

Vibrio vulnificus serovar E (biotype 2) strains are virulent for eels and have also been reported to cause illness in humans. Studies on the plasmid content of serovar E strains revealed the existence of a plasmid of approximately 70 kbp present in most of these strains. In this study we characterized the 70 kbp plasmids of seven biotype 2 strains isolated from seawater, diseased eels and wound infections in humans. We determined the exact size of the high molecular weight plasmids to be 68 kbp. A comparison of the plasmids of the seven strains by restriction length polymorphism and hybridization analysis showed them to be almost identical.

A highly homologous 68 kbp plasmid found inVibrio vulnificus strains virulent for eels

Vibrio vulnificus serovar E (biotype 2) strains are virulent for eels and have also been reported to cause illness in humans. Studies on the plasmid content of serovar E strains revealed the existence of a plasmid of approximately 70 kbp present in most of these strains. In this study we characterized the 70 kbp plasmids of seven biotype 2 strains isolated from seawater, diseased eels and wound infections in humans. We determined the exact size of the high molecular weight plasmids to be 68 kbp. A comparison of the plasmids of the seven strains by restriction length polymorphism and hybridization analysis showed them to be almost identical.

An indirect immunofluorescent antibody technique for detection and enumeration of Vibrio vulnificus serovar E (biotype 2): development and applications.

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.

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.

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.

&lt;I&gt;Vibrio vulnificus&lt;/I&gt;敗血症によって急死した1剖検例

A 66-year-old male with chronic alcoholic liver injury was admitted on July 27, 1986 to our hospital with complaints of high fever, convulsion and skin erythema. He had consumed raw fish 3 days before, and had a scratch wound over the right arm and left leg because he had slipped in a small stream in the woods the day before admission. He was already in shock state with sepsis of V. vulnificus and DIC on admission. Although the treatment with ABPC, CP, CAZ, MINO for sepsis, and Heparin & Antithrombin III for DIC was immediately begun, he died only 10 hours after admission. On autopsy, the skin lesion revealed phlegmon with necrotizing angitis and the liver showed fatty changes with Mallory's body. The causative organism was detected from the blood and on autopsy from the skin wound, bile juice, liver, spleen, kidney and bone marrow, and its type was determined as a V. vulnificus serovar 4. It was suspected that the route of infection in this case was the raw fish rather than via the wound because the water in which he had been wounded was fresh water and the bacterium was not detected from the water, shells, nor moss existing there.