Vibrio Anguillarum O2 Research Articles

Antibacterial Activity of Ulva Lactuca Against Important Aquaculture Bacterial Strains

Efforts are made to produce functional aquaculture diets capable of promoting fish growth and health while being sustainable at social, economic and environmental levels. One of the emerging threats in aquaculture has become the antibiotic resistance phenomena due to antimicrobial drugs. Therefore, functional feed additives of marine origin have been introduced globally as an alternative to fish antibiotics. Towards the selection of natural and sustainable resources of bioactive compounds, seaweed has been proven to be a source of valuable substances showing antibacterial activity. Aim of this study is to evaluate the inhibition of bacterial growth caused by an ethanolic extract of the macroalgae Ulva lactuca against important aquaculture bacterial strains. Vibrio anguillarum O1, Photobacterium damselae sub. piscicida and Tenacibaculum maritimum, were incubated for 48 hours in sterile Brain Heart Infusion Broth and tested for resistance to the extract using broth cultures. The algal extract successfully inhibited the growth of all strains. The optimum inhibition was achieved for VAO1 and PHDP by the undiluted and 1:1 diluted extract, while for TMAR, by the 1:3 dilution as well. The antibacterial activity that the extract provided is considered to be the result of the containing bioactive compounds of the algal strain, such as polysaccharides, carotenoids and phenolic compounds.

The effect of temperature and challenge route on in vitro hemocyte phagocytosis activation after experimental challenge of common octopus, Octopus vulgaris (Cuvier, 1797) with either Photobacterium damselae subsp. damselae or Vibrio anguillarum O1.

Infectious diseases in aquaculture could be associated with high mortalities and morbidity rates, resulting in negative impacts to fish farming industry, consumers, and the environment. Octopods are reared near marine fish farming areas, and this may represent a major risk since fish pathogens may cause pathologies to octopods. Up to date cephalopods immune defense and pathologies, are incompletely understood. Therefore, the aim of this study was to determine the effect of water temperature and challenge route on hemocyte phagocytosis in vitro after experimental challenge of common octopus with Photobacterium damselae subsp. damselae or Vibrio anguillarum O1. Hemolymph was withdrawn at various time-points post-challenge and the number of circulating hemocytes, and phagocytosis ability were determined. No mortalities were recorded irrespective of pathogen, route of challenge and temperature employed. Great variation was observed in the number of circulating hemocytes of both control and challenged specimens in both experiments (1.04×10⁵ to 22.33×10⁵ hemocytes/ml for the Photobacterium damselae subsp. damselae challenge and 1.35×105 to 24.63×105 hemocytes/ml for the Vibrio anguillarum O1 and at both studied temperatures). No correlation was found between circulating hemocytes and baseline control specimens body weight. Probably, the number of circulating hemocytes is affected by many extrinsic, and intrinsic factors such as size, age, maturity stage, natural fluctuations and temperature, as indicated in the literature. The hemocyte foreign particles binding ability observed in Photobacterium damselae subsp. damselae experiments, at 21±0.5°C and 24±0.5°C, was (mean±SD) 2.26±2.96 and 11.72±12.36 yeast cells/hemocyte for baseline specimens and 7.84±8.88 and 8.56±9.89 yeast cells/hemocyte for control and challenged specimens, respectively. The corresponding values for Vibrio anguillarum O1 experiments were (mean±SD) 6.68±9.26 and 7.00±8.11 yeast cells/hemocyte for baseline specimens and 8.82±9.75 and 6.04±7.64 yeast cells/hemocyte for control and challenged specimens, respectively. Hemocytes of the Photobacterium damselae subsp. damselae and Vibrio anguillarum O1 challenged specimens, were more activated at lower temperature. Apparently, temperature is an important factor in hemocyte activation. In addition, our results indicated that time post challenge, route of challenge and pathogen may influence phagocytosis ability.

Evaluation of in vitro and in vivo potential of Bacillus subtilis MBTDCMFRI Ba37 as a candidate probiont in fish health management

Bacillus subtilis MBTDCMFRI Ba37 (B. subtilis Ba37), an antibacterial strain isolated from the tropical estuarine habitats of Cochin, was evaluated for in vitro and in vivo potential, and its application as a candidate probiont in fish health management. B. subtilis Ba37 was characterized using their morphological and biochemical properties. It exhibited exoenzymatic activities, tolerance to various physiological conditions and a wide spectrum of antibacterial activity against aquaculture pathogens such as Vibrio and Aeromonas. In co-culture assay, B. subtilis Ba37 inhibited Vibrio anguillarum O1 (V. anguillarum O1) even with the initial cell count of 104 CFUmL−1. Cytotoxicity assay performed using the cell free supernatant (CFS) of B. subtilis Ba37 revealed its non toxic nature. A twenty one days of feeding trial was conducted in juveniles of Etroplus suratensis (E.suratensis) by administrating B. subtilis Ba37 to evaluate its efficacy on growth, immune parameters and antioxidant enzyme activities. Overall the supplementation of B. subtilis Ba37 enhanced significantly (P < 0.05) the survival rate, weight gain, specific growth (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and feed efficiency (FE) of the fed animals as compared with the control. The immune parameters and antioxidant activities such as total protein, alkaline phosphatase (ALP), superoxide dismutase (SOD) and catalase were also improved significantly (P < 0.05) while serum alanine aminotransferase (SGOT) and serum aspartate aminotransferase (SGPT) activities were decreased slightly than the control. After fifteen days of challenge test, the fish fed with B. subtilis Ba37 showed higher relative percentage survival (RPS) than the control. Thus the study indicated the advantages of B. subtilis Ba37 to be used as a candidate probiont, which could be effectively utilized in managing diseases in aquaculture systems and to improve the health of the host.

Transcriptome-wide mapping of signaling pathways and early immune responses in lumpfish leukocytes upon in vitro bacterial exposure

We performed RNA sequencing, identified components of the immune system and mapped early immune responses of lumpfish (Cyclopterus lumpus) leukocytes following in vitro exposure to the pathogenic bacterium Vibrio anguillarum O1. This is the first characterization of immune molecules in lumpfish at the gene level. In silico analyses revealed that genes encoding proteins involved in pathogen recognition, cell signaling and cytokines in mammals and teleosts are conserved in lumpfish. Unique molecules were also identified. Pathogen recognition components include 13 TLRs, several NLRs and complement factors. Transcriptome-wide analyses of immune responses 6 and 24 hours post bacterial exposure revealed differential expression of 9033 and 15225 genes, respectively. These included TLR5S, IL-1β, IL-8, IL-6, TNFα, IL-17A/F3, IL-17C and several components of the complement system. The data generated will be valuable for comparative studies and make an important basis for further functional analyses of immune and pathogenicity mechanisms. Such knowledge is also important for design of immunoprophylactic measures in lumpfish, a species of fish now farmed intensively for use as cleaner-fish in Atlantic salmon (Salmo salar) aquaculture.

Interaction of a 22 kDa Peptidyl Prolyl cis/trans Isomerase with the Heat Shock Protein DnaK in Vibrio anguillarum.

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds preceding prolines. We investigated the protein-protein interaction between a 22 kDa PPIase (VaFKBP22, an FK506-binding protein) and the molecular chaperone DnaK derived from Vibrio anguillarum O1 (VaDnaK) using GST pull-down assays and a bacterial two-hybrid system for in vivo and in vitro studies, respectively. Furthermore, we analyzed the three-dimensional structure of the protein-protein interaction. Based on our results, VaFKBP22 appears to act as a cochaperone of VaDnaK, and contributes to protein folding and stabilization via its peptidyl-prolyl cis/trans isomerization activity.

Adjuvant Effect of Quillaja saponaria Saponin (QSS) on Protective Efficacy and IgM Generation in Turbot (Scophthalmus maximus) upon Immersion Vaccination.

The adjuvant effect of Quillaja saponaria saponin (QSS) on protection of turbot fry was investigated with immersion vaccination of formalin-killed Vibrio anguillarum O1 and various concentrations of QSS (5, 25, 45 and 65 mg/L). Fish were challenged at days 7, 14 and 28 post-vaccination. Significantly high relative percent of survival (RPS) ((59.1 ± 13.6)%, (81.7 ± 8.2)%, (77.8 ± 9.6)%) were recorded in the fish that received bacterins immersion with QSS at 45 mg/L, which is comparable to the positive control group vaccinated by intraperitoneal injection (IP). Moreover, a remarkably higher serum antibody titer was also demonstrated after 28 days in the vaccinated fish with QSS (45 mg/L) than those vaccinated fish without QSS (p < 0.05), but lower than the IP immunized fish (p < 0.05). Significant upregulation of IgM gene expression has also been identified in the tissues of skin, gill, spleen and kidney from the immunized fish in comparison to the control fish. Taken together, the present study indicated that QSS was able to dramatically evoke systemic and mucosal immune responses in immunized fish. Therefore, QSS might be a promising adjuvant candidate for fish vaccination via an immersion administering route.

Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.

Isolation and characterization of a 17-kDa FKBP-type peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum

Peptidyl-prolyl cis/trans isomerase (PPIase) catalyzes the isomerization of peptide bonds to achieve conformational changes in native folded proteins. An FKBP-type PPIase with an approximate molecular weight of 17kDa was isolated from Vibrio anguillarum O1 and named VaFKBP17. To investigate its biochemical properties, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli. A protease-coupled assay for isomerization activity, using Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate, indicated that the activity of VaFKBP17 was highest at low temperature (5°C) and alkaline conditions (pH 10). The immunosuppressant FK506 inhibited the isomerization activity of VaFKBP17. The chaperone activity of VaFKBP17 was assessed using a citrate synthase thermal aggregation activity assay. To evaluate its ability to catalyze protein refolding, the effect of VaFKBP17 on inclusion bodies was investigated during a dilution process. In this assay, VaFKBP17 was able to assist protein refolding. These results provide evidence that VaFKBP17 possesses chaperone-like activity. The structural homology of VaFKBP17 relative to other known bacterial FKBPs was also examined.

Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

BackgroundDespite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid marker for assessing vaccination status in a fish population. For this purpose a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate sera from farmed rainbow trout vaccinated against V. anguillarum O1.Study DesignImmune sera from rainbow trout immunised with an experimental vaccine based on inactivated V. anguillarum O1 bacterin in Freund’s incomplete adjuvant were used for ELISA optimisation. Subsequently, sera from farmed rainbow trout vaccinated with a commercial vaccine against V. anguillarum were analysed with the ELISA. The measured serum antibody levels were compared with the vaccine status of the fish (vaccinated/unvaccinated) as evaluated through visual examination.ResultsRepeated immunisation with the experimental vaccine lead to increasing levels of specific serum antibodies in the vaccinated rainbow trout. The farmed rainbow trout responded with high antibody levels to a single injection with the commercial vaccine. However, the diversity in responses was more pronounced in the farmed fish. Primary visual examinations for vaccine status in rainbow trout from the commercial farm revealed a large pool of unvaccinated specimens (vaccination failure rate = 20%) among the otherwise vaccinated fish. Through serum analyses using the ELISA in a blinded set-up it was possible to separate samples collected from the farmed rainbow trout into vaccinated and unvaccinated fish.ConclusionsMuch attention has been devoted to development of new and more effective vaccines. Here we present a case from a Danish rainbow trout farm indicating that attention should also be directed to the vaccination procedure in order to secure high vaccination frequencies necessary for optimal protection with a reported effective vaccine.

Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod ( Gadus morhua L.) juveniles

The purpose of this study was to investigate the efficacy of three monovalent and a trivalent vibriosis dip vaccines in juvenile Atlantic cod ( Gadus morhua L.), examine whether the responses were specific and study the expression of selected immune genes after dip vaccination. In addition, the study addressed whether the deviating isolates of Vibrio anguillarum serotype O2 belongs to another sero-subgroup than the previously established sero-subgroups O2a, O2b and O2c. Rabbit V. anguillarum serotype O2 antiserum adsorbed with V. anguillarum O2a O-antigen was shown, by both ELISA and immunoblotting, to still contain serotype O2 specific antibodies. Cod V. anguillarum serotype O2 antiserum reacted only with isolate of homologous serotype and not with heterologous sero-subgroups. This indicates that the deviating V. anguillarum O2 isolates represent a new sero-subgroup differing from sero-subgroup O2a. The monovalent vaccines included formalin inactivated cultures of V. anguillarum sero-subgroup O2a, O2b or serotype O2, while the trivalent vaccine contained all three sero-subgroups. Cod mounted high protection 7 weeks post dip vaccination with monovalent vaccines when challenged with homologous isolates and significantly lower when challenged with heterologous isolates, regardless of sero-subgroups. The trivalent vaccine resulted in efficient protection against all sero-subgroups tested. Dip vaccination of cod juveniles did not result in detectable antibody production or alteration in gene expression of the heavy chain of IgM and IgD. In the trivalent vaccine group expression of IFNγ and IL-12p40 were significantly up-regulated 3 days post vaccination. However, in groups vaccinated against V. anguillarum sero-subgroups O2b or O2, IL-12p40 and IFNγ gene expression were slightly increased 3 and 55 days post vaccination, respectively.

Identification ofVibrio anguillarumOuter Membrane Vesicles Related to Immunostimulation in the Japanese Flounder,Paralichthys olivaceus

We identified outer membrane vesicle (OMV) production in Vibrio anguillarum O1, a major fish pathogen that causes vibriosis, and characterized the OMVs. They were produced during normal growth, and were appeared as spherical vesicle fractions. The protein profile of the OMVs was similar to that of the outer membrane proteins, and the 38-kDa major protein band of OMV was identified as OmpU. The OMVs had enzyme activity of metalloprotease, hemolysin, and phospholipase, and stimulated the production of proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 when injected into the flounder.

Vibriosis as an Epizootic Disease of Rainbow Trout (Onchorynchus mykiss) in Turkey

Vibrio anguillarum O1 was isolated from diseased cultured rainbow trout Oncorhynchus mykiss, in the Southern Aegean of Turkey with lesions and signs of vibriosis. Gram (-), motile and curved rods from diseased rainbow trout were identified after physiological, biochemical and slide agglutination tests as V. anguillarum O1. Koch postulates were fulfilled with the isolated bacteria. The antibiotic susceptible test results showed that V. anguillarum strains were susceptibility to amoxycillin, ampycilline, flumequin, furazolidone, kanamycin, potentiated sulphonamide and oxolinic acid.

Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes

The aim of this study was to investigate the possibility of mucosal vaccination in African catfish ( Clarias gariepinus) with Vibrio anguillarum O2 bacterins. The antigen was administered via different routes: anal intubation, oral administration, intraperitoneal injection and immersion. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Increased antibody levels were found in bile and mucus upon anal intubation, which was not the case after intraperitoneal injection. The data indicate that oral vaccination of fish may be possible when antigens can reach the second gut segment in sufficient quantities and in the right form as confirmed by the recorded substantial induction of systemic and mucosal immunity. The results obtained are a strong indication for mucosal immune response and the two compartmental models for immune response in fish.

High-M alginate immunostimulation of Atlantic halibut ( Hippoglossus hippoglossus L.) larvae using Artemia for delivery, increases resistance against vibriosis

A strategy for microbial management in marine juvenile production should include nonspecific immunostimulation to reduce losses caused by infectious diseases. Halibut ( Hippoglossus hippoglossus L.) larvae (250 day degrees) were distributed into first-feeding tanks of 10.000 l, at a Norwegian halibut juvenile farm. Immunomodulatory high-M alginate was bioencapsulated in Artemia by use of alginate microbeads (2–30 μm), and administered to halibut larvae at three stages during the first feeding period (days 7–9, 20–22 and 41–43). A fourth treatment was run after weaning, 5–3 days prior to a challenge test with two different doses of Vibrio anguillarum O2 that was started at day 90 after first feeding. After 15 days, the mortality at the highest dose was 45±1% in the control and 28±8% in the stimulated group, corresponding to 38% reduction in mortality, whereas no significant differences were measured at the low dose. It is concluded that feeding immunostimulants to halibut larvae for four short periods during 3 months enhanced resistance against vibriosis, and that high-M alginates are suitable as immunostimulants for marine larvae during first feeding and weaning.

Vaccination of African catfish with Vibrio anguillarum O2: II. Oral intubation with vaccine-containing pellets

The aim of this study was to investigate the success of oral vaccination application in African catfish using Vibrio anguillarum O2 bacterins. The antigen uptake was followed by competitive enzyme-linked immunosorbent assay (ELISA). Serum antibody response was measured using an indirect ELISA. Several in vivo administration methods were investigated. Intraperitoneal injection gave the highest absorption rate, with high antibody levels in the systemic circulation. Oral intubation of bacterin-layered pellets resulted in low antigen uptake and low antibody levels. The addition of absorption enhancers increased the serum antigen levels. An enteric coating applied on the pellets containing vaccine did not improve the immune response.

Vaccination of African catfish with Vibrio anguillarum O2: I. ELISA development and response to IP and immersion vaccination

The aim of this study was to investigate African catfish (Clarias gariepinus) vaccination with Vibrio anguillarum O2 bacterins. To monitor the antigen uptake, a competitive enzyme-linked immunosorbent assay (ELISA) was developed. Serum antibody response was measured using an indirect ELISA. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblot data that characterize the immunoglobulin molecule of the African catfish are presented. The impact of acid conditions on the antigen proved to be stable above pH 4. A partition coefficient was calculated to determine the ability of transepithelial uptake. Two in vivo administration methods were investigated in the study. Intraperitoneal injection gave the highest absorption rate with high antibody levels in the systemic circulation, whereas immersion did not induce significant serum antibody levels.

Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets

The impact on antigen uptake and antibody response by the addition of absorption enhancers to Vibrio anguillarum O2 antigen was studied in oral vaccination trials of African catfish ( Clarias gariepinus). Oral vaccination was achieved by feeding lag time coated pellets. The lag time coat prevents premature release of the encapsulated vaccine in the tank, before ingestion of the pellets by the fish. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Feeding of bacterin-layered pellets without absorption enhancers resulted in a rather low antigen uptake and antibody levels. The addition of absorption enhancers such as sodium salicylate, sodium caprate and vitamin E TPGS increased the serum antigen levels and specific antibody levels in the systemic circulation. Skin mucus antibody levels were higher after oral vaccination compared to the IP and control group. The addition of absorption enhancers in the oral groups further increased the antibody levels obtained in the skin mucus.

Genetic organization of the regions associated with surface polysaccharide synthesis in Vibrio cholerae O1, O139 and Vibrio anguillarum O1 and O2: a review.

Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by.

Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined.

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens