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 …
The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels
Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans. Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E). In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 str…
MARTX Toxin in the Zoonotic Serovar of Vibrio vulnificus Triggers an Early Cytokine Storm in Mice
Vibrio vulnificus biotype 2-serovar E is a zoonotic clonal complex that can cause death by sepsis in humans and fish. Unlike other biotypes, Bt2 produces a unique type of MARTXVv (Multifunctional-Autoprocessive-Repeats-in-Toxin; RtxA13), which is encoded by a gene duplicated in the pVvBt2 plasmid and chromosome II. In this work, we analyzed the activity of this toxin and its role in human sepsis by performing in vitro, ex vivo, and in vivo assays. First, we demonstrated that the ACD domain, present exclusively in this toxin variant, effectively has an actin-cross-linking activity. Second, we determined that the whole toxin caused death of human endotheliocytes and monocytes by lysis and apo…
The Effect of the Environmental Temperature on the Adaptation to Host in the Zoonotic Pathogen Vibrio vulnificus
Vibrio vulnificus is a zoonotic pathogen that lives in temperate, tropical and subtropical aquatic ecosystems whose geographical distribution is expanding due to global warming. The species is genetically variable and only the strains that belong to the zoonotic clonal-complex can cause vibriosis in both humans and fish (being its main host the eel). Interestingly, the severity of the vibriosis in the eel and the human depends largely on the water temperature (highly virulent at 28°C, avirulent at 20°C or below) and on the iron content in the blood, respectively. The objective of this work was to unravel the role of temperature in the adaptation to the host through a transcriptomic and phen…
Bottlenecks in diagnostics of mediterranean fish diseases
Three independent Horizon 2020 projects are prioritising a common task of solving diagnostic issues using different approaches and strategies. PerformFISH focuses its research objectives on diagnostic methods, MedAID has prepared a diagnostic manual for the most devastating fish diseases of the Mediterranean and ParaFishControl has prepared a diagnostic manual on parasitic diseases of European aquaculture species. Currently, the projects are investing large collaborative efforts to reinforce their tasks and to deliver mutual and cutting-edge material on the subject. Nevertheless, further efforts for the harmonisation, standardisation and validation of diagnostic methods for infectious disea…
Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels
Vibrio vulnificus biotype 2 is classically considered an obligate eel pathogen. However, it has recently been associated with one human septicemic case. In this paper, the opportunistic behavior of this pathogen is discussed. The bacterium can survive alone in brackish water or attached to eel surfaces for at least 14 days. It is able to spread through water and infect healthy eels by using skin as a portal of entry. These results suggest that water and infected eels may act as reservoirs of infection. A capsule seems to be essential for waterborne infectivity, which would explain why cells recovered from naturally diseased eels give rise to pure cultures of opaque colonies. The spread of t…
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 constit…
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) CEC…
Susceptibility of Nile tilapia (Oreochromis niloticus) to vibriosis due to Vibrio vulnificus biotype 2 (serovar E)
The present study documents the susceptibility of Nile tilapia to the experimental vibriosis caused by Vibrio vulnificus biotype 2 (serovar E) using a reference strain (Spanish Collection of Type Cultures, CECT 4604) selected for its high degree of virulence for eels. The biotype 1 of this species is one of the usual organisms involved in epizootics occurred in tilapia. After intraperitoneal injection, the selected strain developed a haemorrhagic septicaemia similar to eel vibriosis with a LD50 four log units lower than that exhibited by the type strain of the species, which belongs to the biotype 1. The results obtained in waterborne and intubation challenges indicated that water and feed …
Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla
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 activ…
Transmission to Eels, Portals of Entry, and Putative Reservoirs of Vibrio vulnificus Serovar E (Biotype 2)
ABSTRACT 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…
Role of iron in the pathogenicity of Vibrio damsela for fish and mammals.
The ability to obtain iron of 14 isolates of Vibrio damsela with different degrees of virulence for mice and turbot (Scophthalmus maximus) has been evaluated in artificial and natural iron-restricted environments. All strains were capable of utilizing haemoglobin (Hb) and ferric ammonium citrate (FAC) as the sole iron sources in vitro. However, only virulent V. damsela strains were able to resist the bacteriostatic and bactericidal effects of human and turbot sera, their growth being enhanced by the addition of Hb and FAC. The inhibitory effect of these sera on the growth of the non-pathogenic strain (ATCC 35083), however, was reversed by heat treatment (56 degrees C for 60 min). The role o…
Survival of fish-virulent strains ofPhotobacterium damselaesubsp.damselaein seawater under starvation conditions
The survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater microcosms, with and without sediment, was investigated. The strains survived as culturable bacteria at 14 and 22 degrees C for at least 1 year, and infectivity for fish was maintained. At 5 degrees C, cells lost culturability on solid media, but this was recovered when the temperature was increased to 22 degrees C. Finally, morphological changes in the bacterium (rod to coccus), and production of vesicles and extracellular material were observed during the time of starvation. The overall results suggest that seawater and sediment can act as reservoirs for these virulent strains.
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…
Biochemical and molecular characterization of three serologically different Vibrio harveyi strains isolated from farmed Dicentrarchus labrax from the Adriatic Sea.
AbstractVibrio harveyi is recognized as one of the major causes of vibriosis, a disease that threatens the long-term sustainability of aquaculture. Current research shows that the Mediterranean strains of V. harveyi are serologically heterogeneous, though research comparing the traits of different strains is scarce. This study aims to describe the biochemical, physiological and genetic characteristics of three serologically different strains of V. harveyi isolated from farmed European Sea bass (Dicentrarchus labrax) from the Adriatic Sea. A total of 32 morphological and biochemical markers were examined and, the susceptibility to 13 antimicrobials tested, and then compared the results of hi…
Ferric-reductase activities in Vibrio vulnificus biotypes 1 and 2.
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 …
Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2
Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans. In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis. The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels. This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation. No alterations in lipopolysaccharide …
Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae
The transmission through water of the disease caused by the fish pathogen, Photobacterium damselae subsp. damselae, as well as the role of the skin mucus in the initial steps of the infection, have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those virulent by the intraperitoneal route were infective through water. Moribund fishes showed the typical signs of the disease: haemorrhaged areas on the body surface and ulcerative lesions with mucus degradation. These results suggest that the pathogen can be transmitted to fish through water and use the skin as a portal of entry.
Evaluation of genotypic and phenotypic methods to distinguish clinical from environmental Vibrio vulnificus strains.
ABSTRACT Vibrio vulnificus is a heterogeneous bacterial species that comprises virulent and avirulent strains from environmental and clinical sources that have been grouped into three biotypes. To validate the typing methods proposed to distinguish clinical from environmental isolates, we performed phenotypic (API 20E, API 20NE, and BIOLOG tests) and genetic (ribotyping and DNA polymorphism at several loci) studies with a large strain collection representing different biotypes, origins, and host ranges. No phenotypic method was useful for biotyping or grouping strains with regard to the origin of an isolate, and only the BIOLOG system was reliable for identifying the strains at the species …
Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
[Background]: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term tr…
Pyrosequencing survey of intestinal microbiota diversity in cultured sea bass (Dicentrarchus labrax) fed functional diets.
The routine use of chemotherapy to control bacterial diseases in aquatic populations has resulted in the development and spread of antibiotic resistance. The inclusion of immunostimulants in fish diets (functional diets) is one of the main strategies to solve this threat. This study aimed to analyse the intestinal microbiota of cultured European sea bass (Dicentrarchus labrax) fed two functional diets applying pyrosequencing of PCR-amplified 16S rRNA gene. Quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. The autochthonous intestinal microbiota of sea bass consisted of two dominant bacterial genera: Dysgonomonas (Bacte…
Additional file 7: Table S2. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
List of proteins detected in anterior and posterior intestine pooled samples. Data on protein expression are mean ± SEM of 4 pools fed the experimental diets. The number of the contig in the Sea Bream Database ( http://nutrigroup-iats.org/seabreamdb ) is indicated. (PDF 1435 kb)
Efficacy of oral reimmunisation after immersion vaccination against Vibrio vulnificus in farmed European eels
Abstract Vulnivaccine, a licensed vaccine against Vibrio vulnificus , protects eels against vibriosis after vaccination by triple prolonged immersion at glass eel stage. Protection lasts for at least 6 months, but later, protection decreases, and eels can suffer stress-related vibriosis. The main objective of this work was to design an oral vaccine that can be used for reimmunisation at any developmental eel stage. With this aim, the efficacy of Vulnivaccine mixed with food as an oral booster was tested in an eel farm. The protection and the immune response (innate and acquired) in serum, mucus (from skin and intestine) and bile were evaluated in reimmunised and control animals (non-reimmun…
Immunomodulatory effects of dietary β-1,3-glucan from Euglena gracilis in rainbow trout (Oncorhynchus mykiss) immersion vaccinated against Yersinia ruckeri
Abstract Potential immunostimulatory effects of orally administered β-glucan were investigated in combination with immersion vaccination against enteric redmouth disease caused by Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). A linear, unbranched and pure (purity ≥98%) β-1,3-glucan (syn. paramylon) from the alga Euglena gracilis was applied at an inclusion level of 1% β-glucan in feed administered at a rate of 1% biomass day−1 for 84 consecutive days. Fish were vaccinated after two weeks of experimental feeding and bath challenged with live Y. ruckeri six weeks post-vaccination. Blood and head kidney were sampled at day 0, 13 (1 day pre-vaccination), 15, 55, 59 (day 3 post-challe…
Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain
Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have be…
Implementation of Antibiotic Discovery by Student Crowdsourcing in the Valencian Community Through a Service Learning Strategy
© 2020 Maicas, Fouz, Figàs-Segura, Zueco, Rico, Navarro, Carbó, Segura-García and Biosca.
Additional file 7: Table S2. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
List of proteins detected in anterior and posterior intestine pooled samples. Data on protein expression are mean ± SEM of 4 pools fed the experimental diets. The number of the contig in the Sea Bream Database ( http://nutrigroup-iats.org/seabreamdb ) is indicated. (PDF 1435 kb)
Draft genome sequences of Vibrio vulnificus strains recovered from moribund tilapia
Potentially zoonotic Vibrio vulnificus strains were isolated from vibriosis outbreaks occurring on eastern Mediterranean tilapia farms between 2016 and 2019. In this work, the draft genome sequences of three representative isolates are presented.
Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice
The virulence mechanisms of Vibrio vulnificus biotype 2 have been studied and compared with those of biotype 1 in mice as the experimental animals. Biotype 2 isolates from European eels were as virulent for mice as biotype 1 strains (50% lethal dose, about 10(5) CFU per mouse); a septicemic infection developed in less than 24 h. These strains had several properties in common with biotype 1 organisms including capsule expression, uptake of various iron sources, and production of exoproteins, whose role in mouse virulence has been demonstrated. We also discuss the implication of biotype 2 strains in human infections.
Additional file 8: Table S3. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
List of proteins detected in anterior intestine samples. Data on protein expression are mean ± SEM of 6 fish fed the experimental diets. The number of the contig in the Sea Bream Database ( http://nutrigroup-iats.org/seabreamdb ) is indicated. (PDF 562 kb)
Additional file 9: Table S4. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
List of differentially expressed proteins (P
Spontaneous Quinolone Resistance in the Zoonotic Serovar of Vibrio vulnificus
ABSTRACT This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.
Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy
Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). LI comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. LII is linked to the aquaculture industry and incl…
Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2
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 th…
Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality.
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…
The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains: comparison of nucleotide sequences and application to the genetic grouping.
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 lect…
Isolation of a new serovar of Vibrio vulnificus pathogenic for eels cultured in freshwater farms
The present work reports the isolation of a new serovar of Vibrio vulnificus pathogenic for eels cultured in freshwater farms. This serovar, named Serovar A, produced two outbreaks of low morbidity and mortality in one Spanish eel farm in September 2000 and March 2001. The vibriosis affected healthy eels, weighting between 10 and 100 g, that had been vaccinated against the serovar E of the species (formerly biotype 2) at least 6 months before. Some of the diseased eels showed symptoms different to those reported for the classical vibriosis due to serovar E. These symptoms included bleeding ventral ulcers together with haemorrhagic intestine. Bath experimental challenges demonstrated that (i…
Isolation of a hemin and hemoglobin binding outer membrane protein of Vibrio vulnificus biotype 2 (serogroup E)
The eel pathogen Vibrio vulnificus biotype 2 (serogroup E) is able to use hemin (Hm) or hemoglobin (Hb) as the sole iron source for growth in vitro and in vivo. The mechanism of heme-iron acquisition in this bacterium requires a direct interaction through binding sites on the bacterial surface (constitutive outer membrane proteins). Using affinity chromatography techniques, a unique protein of around 36.5 kDa was isolated from cell envelopes of E86 strain regardless of the affinity ligand used, hemoglobin or hemin. This protein was purified from both iron-enriched and iron-restricted grown cells. These results support the hypothesis that in this pathogen Hm- and Hb-iron acquisition is media…
Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy
First description of non-motile Yersinia ruckeri serovar I strains causing disease in rainbow trout, Oncorhynchus mykiss (Walbaum), cultured in Spain.
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) …
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 te…
A multiplex PCR for the detection of Vibrio vulnificus hazardous to human and/or animal health from seafood
Vibrio vulnificus is a zoonotic pathogen linked to aquaculture that is spreading due to climate change. The pathogen can be transmitted to humans and animals by ingestion of raw shellfish or seafood feed, respectively. The aim of this work was to design and test a new procedure to detect V. vulnificus hazardous to human and/or animal health in food/feed samples. For this purpose, we combined a pre-enrichment step with multiplex PCR using primers for the species and for human and animal virulence markers. In vitro assays with mixed DNA from different Vibrio species and Vibrio cultures showed that the new protocol was 100 % specific with a detection limit of 10 cfu/mL. The protocol was succes…
Isolation of Vibrio vulnificus Serovar E from Aquatic Habitats in Taiwan
ABSTRACT 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 Hin dIII 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.
Gut microbiota changes in rainbow trout, Oncorhynchus mykiss (Walbaum), during organic acid feed supplementation and Yersinia ruckeri infection
Gut microbiota (GM) composition of vertebrates are considered to influence health parameters including immunological parameters in the host but knowledge of these issues in fish are sparse. We have performed a 45 day experimental study elucidating effects of dietary supplementation of organic acids (Mera Cid) and immunostimulants on the GM composition of rainbow trout Oncorhynchus mykiss (Walbaum) and investigated its association with blood parameters and disease resistance (against enteric red mouth disease (ERM) caused the bacterium Yersinia ruckeri). A total of 640 fish received either 1) basic fish feed without organic acids or immunostimulants, 2) fish feed with immunostimulants (β6glu…
A comparative epizootiologic study of the two fish-pathogenic serovars ofVibrio vulnificusbiotype 2
Vibrio vulnificus biotype 2 is subdivided into two main serovars, serovar E, able to infect fish and humans, and serovar A, only virulent for fish. Serovar E emerged in 1976 as the causative agent of a haemorrhagic septicaemia (warm-water vibriosis) affecting eels cultured in brackish water. Serovar A emerged in 2000 in freshwater-cultured eels vaccinated against serovar E, causing warm-water vibriosis with fish showing a haemorrhagic intestine as the main differential sign. The aim of the present work was to compare the disease caused by both serovars in terms of transmission routes, portals of entry and host range. Results of bath, patch-contact and oral-anal challenges demonstrated that …
Additional file 9: Table S4. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
List of differentially expressed proteins (P
Toxicity of the extracellular products ofVibrio damsela isolated from diseased fish
In this work we analyzed the pathogenic in vivo and in vitro activities for both fish and mammals of extracellular products (ECP) of several isolates of Vibrio damsela implicated in disease problems in marine culture. The ECP from all the strains were strongly lethal for fish (LD50 ranging from 0.06 to 3.7 μg protein/g fish) and mice (LD50 ranging from 0.02 to 0.43 μg protein/g mouse), causing death between 4 and 72 h after inoculation. These ECP samples possessed low proteolytic activity without production of caseinase, gelatinase, or elastase. However, most of them showed remarkable phospholipase and hemolytic activity for sheep, human, and turbot red blood cells. In addition, all the ECP…
Additional file 8: Table S3. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
List of proteins detected in anterior intestine samples. Data on protein expression are mean ± SEM of 6 fish fed the experimental diets. The number of the contig in the Sea Bream Database ( http://nutrigroup-iats.org/seabreamdb ) is indicated. (PDF 562 kb)
Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains of Vibrio vulnificus biotypes 1 and 2 by silver staining and immunoblotting.
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,…
High affinity iron-uptake systems in Vibrio damsela: role in the acquisition of iron from transferrin
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 resp…
The Fish Pathogen Vibrio vulnificus Biotype 2: Epidemiology, Phylogeny, and Virulence Factors Involved in Warm-Water Vibriosis
ABSTRACT Vibrio vulnificus biotype 2 is the etiological agent of warm-water vibriosis, a disease that affects eels and other teleosts, especially in fish farms. Biotype 2 is polyphyletic and probably emerged from aquatic bacteria by acquisition of a transferable virulence plasmid that encodes resistance to innate immunity of eels and other teleosts. Interestingly, biotype 2 comprises a zoonotic clonal complex designated as serovar E that has extended worldwide. One of the most interesting virulence factors produced by serovar E is RtxA1 3 , a multifunctional protein that acts as a lethal factor for fish, an invasion factor for mice, and a survival factor outside the host. Two practically id…
The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms
Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, ge…
Additional file 3: Figure S2. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona visualisation of the relative abundance of intestinal bacterial OTUs identified in fish fed D1. (HTML 238 kb)
Additional file 2: Table S1. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Percentage of the different bacterial OTUs identified in intestinal mucus of fish fed different diets. The results can be sorted by Phylum, Family, Genus and Species (OTUs). Numbers represent the mean percentage of four replicate pools. (XLSX 63 kb)
The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms
Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, ge…
Additional file 6: Figure S5. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona analysis of the relative abundance of intestinal bacterial OTUs identified in fish fed D4. (HTML 250 kb)
Additional file 6: Figure S5. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona analysis of the relative abundance of intestinal bacterial OTUs identified in fish fed D4. (HTML 250 kb)
Additional file 2: Table S1. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Percentage of the different bacterial OTUs identified in intestinal mucus of fish fed different diets. The results can be sorted by Phylum, Family, Genus and Species (OTUs). Numbers represent the mean percentage of four replicate pools. (XLSX 63 kb)
Additional file 4: Figure S3. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona analysis of the relative abundance of intestinal bacterial OTUs identified in fish fed D2. (HTML 243 kb)
Additional file 5: Figure S4. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona analysis of the relative abundance of intestinal bacterial OTUs identified in fish fed D3. (HTML 246 kb)
Additional file 1: Figure S1. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Rarefaction curves obtained from the sequencing data of the four different pooled samples per dietary group. (TIFF 437 kb)
Additional file 4: Figure S3. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona analysis of the relative abundance of intestinal bacterial OTUs identified in fish fed D2. (HTML 243 kb)
Additional file 5: Figure S4. of Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets
Krona analysis of the relative abundance of intestinal bacterial OTUs identified in fish fed D3. (HTML 246 kb)