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A mathematical first-order difference equation was designed to predict the dynamics of the phage-bacterium adsorption process in aquatic environments, under laboratory conditions. Our model requires knowledge of bacteria and bacteriophage concentrations and the measurements of bacterial size and velocity to predict both the number of bacteriophages adsorbed onto their bacterial host and the number of infected bacteria in a given specific time. It does not require data from previously performed adhesion experiments. The predictions generated by our model were validated in laboratory. Our model was initially conceived as an estimator for the effectiveness of the inoculation of phages as antibacterial therapy for aquaculture, is also suitable for a wide range of potential applications.

Graphical abstract, This work details a protocol for recovering bacteriophages from intertidal sessile mussels and testing their lytic activity against pathogenic bacteria. Although bacteriophages were highly abundant in coastal filter-feeding organisms, they were not detectable in the surrounding water column. This difference reflects the high filtering rate of the mussels, which capture and concentrate high amounts of bacteria, generating an ideal environment for bacteriophages. We validated the protocol providing a mean concentration of 4E + 04 PFU mL-1 lytic bacteriophages for the fish-pathogen bacterium Vibrio ordalii. We suggest that this method has particular utility for the recovery of bacteriophages for use as natural antimicrobial agents in aquaculture.

Vibriosis outbreaks due to Vibrio ordalii occur globally, but Chilean salmon aquaculture, in particular, has suffered significant monetary losses in the last 15 years. Little is known about the virulence mechanisms employed by V. ordalii. However, most Vibrio pathogens (e.g., Vibrio anguillarum, a very close taxonomic species) present outer membrane vesicles (OMVs) that are released extracellularly and implicated in the delivery of virulence factors to host cells. This study provides the first reported evidence of the fish pathogen V. ordalii producing and releasing OMVs under normal growth conditions. Analyses were conducted with the V. ordalii strain Vo-LM-18 and the type strain ATCC 33509T . For comparative purposes, the reference strain V. anguillarum ATCC 43307 was employed. The average size for the three Vibrio strains was 0.215 ± 0.6 µm (via scanning electron microscopy) or between 0.19 and 1.8 µm (via dynamic light scattering), with each bacterium presenting a wide range. SDS-PAGE revealed similarities in OMV patterns, but neither total nor external proteins were identical. Comparing V. ordalii ATCC 33509T and Vo-LM-18, bands were most evident in the total proteins, and the greatest degree of similarity in OMV profiles was between 37 and 50 kDa. The purified OMVs demonstrated haemolytic enzyme activity, which could play a role during V. ordalii infection. These data represent an initial step towards gaining new insights into this virulence factor, of which a lot is known in other pathogenic microorganisms.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using &beta, galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen-2 is a commercial polyvalent bath vaccine that contains formalin-inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen-2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath-immunized, bath-boost-immunized at four weeks post-primary immunization, and intraperitoneally (i.p.) boost-immunized at eight weeks post-primary immunization. The control groups were i.p. mock-immunized with PBS. Twenty-seven weeks post-primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD50 dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post-challenge. Commercial vaccine Vibrogen-2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.

Recent studies have shown that marine algae represent a great source of natural compounds with several properties. The lipidic extract of the seaweed Chaetomorpha linum (Chlorophyta, Cladophorales), one of the dominant species in the Mar Piccolo of Taranto (Mediterranean, Ionian Sea), revealed an antibacterial activity against Vibrio ordalii and Vibrio vulnificus, common pathogens in aquaculture, suggesting its potential employment to control fish and shellfish diseases due to vibriosis and to reduce the public health hazards related to antibiotic use in aquaculture. This extract showed also an antioxidant activity, corresponding to 170.960 &plusmn, 16. mmol Trolox equivalent/g (oxygen radical absorbance capacity assay&mdash, ORAC) and to 30.554 &plusmn, 2.30 mmol Trolox equivalent/g (Trolox equivalent antioxidant capacity assay&mdash, TEAC). The chemical characterization of the extract, performed by 1D and 2D NMR spectroscopy, highlighted the presence of free, saturated (SAFAs), unsaturated (UFAs) and polyunsaturated (PUFAs) fatty acids. The high content of &omega, 6 and &omega, 3 PUFAs confirmed also by gas chromatography indicates the potentiality of this algal species in the production of fortified food. The antibacterial activity seems related to the presence of linolenic acid present at high density, while the antioxidant activity could be likely ascribable to molecules such as carotenoids and chlorophylls (characterized also by thin-layer chromatography), known for this property. The presence of polyhydroxybutyrate, a biopolymer with potentiality in the field of biodegradable bioplastics was also detected. The exploitation of C. linum for a future biotechnological application is also encouraged by the results from a first attempt of cultivating this species in an integrated multi-trophic aquaculture (IMTA) system.

In mariculture, diseases of microbial origin can cause significant economic losses worldwide; the evolution of microorganism resistance to antibiotics has resulted in a growing need for new antibacterial compounds that are effective in veterinary medicine and characterized by limited undesirable side effects. Increased attention has recently been turned to seaweeds as a promising source for metabolites with antimicrobial activity. Vibriosis is a common disease, caused by bacteria of the genus Vibrio, that can result in high mortality in aquaculture. The aim of this study was to identify seaweeds with antibacterial activity against some pathogenic Vibrio species, in order to identify a possible alternative to the commonly used antibiotics in aquaculture. Chloroform/methanol lipidic extracts of six seaweed species (Chaetomorpha linum, Cladophora rupestris, Gracilaria dura, Gracilaria gracilis, Gracilariopsis longissima, Ulva prolifera) were tested for their antibacterial activities against six fish pathogenic Vibrio species using the disc diffusion method. Different susceptibilities to lipidic algal extracts were observed. All six of the seaweed extracts tested demonstrated inhibition of Vibrio ordalii. The best was that from Gracilariopsis longissima, showing activity against Vibrio ordalii, Vibrio salmonicida, Vibrio alginolyticus and Vibrio vulnificus. The results confirmed the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting feed for aquaculture.

The genetic heterogeneity of the close relatives Vibrio anguillarum and Vibrio ordalii , both serious pathogens of fish causing extensive losses in aquaculture, was studied. Eight housekeeping genes, i.e., atpA , ftsZ , gapA , gyrB , mreB , rpoA , topA , and pyrH , were partially sequenced in 116 isolates from diverse fish species and geographical areas. The eight genes appear to be under purifying selection, and the genetic diversity in the total data set was estimated to be 0.767 ± 0.026. Our multilocus sequence analysis (MLSA) scheme identified several widespread clonal complexes and resolved the isolates, for the most part, according to serotype. Serotype O2b isolates from diseased cod in Norway, Ireland, and Scotland were found to be extremely homogeneous. Horizontal gene transfer appears to be fairly common within and between clonal complexes. Taken together, MLSA and in silico DNA-DNA hybridization (DDH) calculations suggest that some isolates previously characterized as V . ordalii , i.e., 12B09, FF93, FS144, and FS238, are in fact V. anguillarum isolates. The precise taxonomic situation for two isolates from Atlantic cod that display several traits consistent with V. ordalii , i.e., NVI 5286 and NVI 5918, and a single environmental strain that was previously considered to represent V. ordalii , i.e., FF167, is less clear. IMPORTANCE It is still being debated whether V. anguillarum and V . ordalii represent separate bacterial species. Our study addresses this issue and elucidates the degree of genetic variability within this group of closely related bacteria, based on a substantial number of isolates. Our results clearly illustrate the existence of different populations among putative V . ordalii isolates. On the basis of additional full-length genomic analysis, we conclude that most environmental isolates previously identified as V . ordalii lie firmly within the species V. anguillarum . While bona fide fish-pathogenic V . ordalii isolates display a very close genetic relationship with V. anguillarum , they combine a clearly divergent evolutionary pattern with clear phenotypic differences. The study also highlights the need for further characterization of fish-pathogenic isolates from the northern Atlantic region that share phenotypic characteristics with V. ordalii but are genetically closer to V. anguillarum . The retention of taxonomic distinctions between the phenotypically different groups of bacteria is of practical advantage to microbial ecologists and veterinarians.

Copepods are abundant crustaceans that harbor diverse bacterial communities, yet the nature of their interactions with microbiota are poorly understood. Here, we report that Vibrio elicits targeted transcriptional responses in the estuarine copepod Eurytemora affinis We pre-treated E. affinis with an antibiotic cocktail and exposed them to either a zooplankton specialist (Vibrio sp. F10 9ZB36) or a free-living species (Vibrio ordalii 12B09) for 24 h. We then identified via RNA-Seq a total of 78 genes that were differentially expressed following Vibrio exposure, including homologs of C-type lectins, chitin-binding proteins and saposins. The response differed between the two Vibrio treatments, with the greatest changes elicited upon inoculation with V. sp. F10 We suggest that these differentially regulated genes play important roles in cuticle integrity, the innate immune response, and general stress response, and that their expression may enable E. affinis to recognize and regulate symbiotic vibrios. We further report that V. sp. F10 culturability is specifically altered upon colonization of E. affinis These findings suggest that rather than acting as passive environmental vectors, copepods discriminately interact with vibrios, which may ultimately impact the abundance and activity of copepod-associated bacteria.

Vibrio ordalii is the causative agent of vibriosis in several cultured salmonid species worldwide. Despite its impact on aquaculture, relatively little information is available about its virulence factors. The present study demonstrates for the first time that V. ordalii possesses different systems of iron acquisition, one involving siderophore synthesis and another one that uses direct binding of heme to use iron. Using 6 strains of V. ordalii from Atlantic salmon Salmo salar and the V. ordalii type strain, we could demonstrate that all strains could grow in presence of the chelating agent 2,2'-dipyridyl and produced siderophores in solid and liquid media. Cross-feeding assays among V. ordalii strains evidenced variability in the siderophores produced. Bioassays and PCR data suggest that V. ordalii could produce a siderophore with a structure similar to piscibactin, although the production of a second siderophore in certain strains cannot be discarded. Furthermore, all strains were able to use hemin and hemoglobin as the only iron sources, although the cell yield was higher when using hemoglobin. A hemin-binding assay indicated the presence of constitutive heme-binding molecules at the cell surface of V. ordalii. Virulence tests using rainbow trout as a model of infection revealed a clear relationship between iron-uptake ability and pathogenicity in V. ordalii.

The aquaculture of the sea cucumber Apostichopus japonicus has grown rapidly over recent years in China and become an important sector of the marine industry. However, with rapid growth and the use of non-standard practices and culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. Prevention and control of diseases seriously affecting aquaculture of A. japonicus , a major marine industry in China, are essential for its development. We have previously isolated two pathogenic bacteria Pseudoalteromonas sp and Pseudoalteromonas tetraodonis associated with ulceration and peristome tumescence of A. japonicus . In this study, we isolated 266 bacteria from A. japonicus and its cultural environment, and identified 5 bacteria Vibrio ordalii, Bacillus safensis, P. tetraodonis, Pseudoalteromonas elyakovii and Pseudomonas segetis with strong antimicrobial activity to P. sp and P. tetraodonis using point inoculation, filter paper and Oxford cylinder methods. Further investigation on their growth and antagonistic characteristics indicated that 1) V. ordalii has the strongest antimicrobial ability even at low temperature and its extracellular products precipitated with ammonium sulfate at different growth stages also have antimicrobial activity; 2) P. tetraodonis and P. elyakovii only have antimicrobial activity at 28 °C and their extracellular products at different growth stages also have strong antagonistic effects; 3) the extracellular products of B. safensis at different growth stages have no antibacterial activity. Injection of extracellular products of P. elyakovii and V. ordalii into A. japonicus can enhance its non-specific immune-related enzyme activities and disease resistance. The study provides a theoretical basis and products for A. japonicus disease prevention and healthy aquaculture.

Forty natural plants collected in Jeju, Jeonnam-Goheung, and Gyeongbuk-Ulleung were extracted using a pressurized liquid. Extraction yields of total soluble solids and total phenolics (TP), and integral antioxidative capacity (IAC) were measured, and antimicrobial activity was tested against Streptococcus parauberis, Streptococcus iniae, Edwardsiella tarda, and Vibrio ordalii. Jipsinnamul showed the highest content of TP (174.4 ㎎ GAE/g), followed by Mulchamnamu (116.9), Seoeonamu (113.3), and Buknamu (108.2). Percent TP/TSS was high in Jipsinnamul (72.6%), Seoeonamu (47.3%), Mulchamnamu (46.4%), Jageumu (40.2%), and Baneulkkot (40.1%), respectively. Magamok, Nadosongipul, Buknamu, Mulchamnamu, and Seoeonamu showed 5.81, 3.96, 3.63, 3.63, and 3.34 mmol ascorbic acid equivalents/g of IAC of water-soluble substances, and Seoeonamu, Magamok, Seipijilpul, Mulchamnamu, Baneulkkot, and Seomgirincho showed 8.51, 6.57, 5.68, 3.85, 3.83, and 3.69 mmol Trolox equivalents/g of IAC of lipid-soluble substances, respectively. Only nine species such as Baneulkkot, Dokhwal, Jipsinnamul, Mulchamnamu, Nadosongipul, Seipijilpul, Seoeonamu, Seomgirincho, and Sumbadi of 40 selected plants showed the antimicrobial activity against four bacteria tested. Jipsinnamul showed the strong antimicrobial activity against S. iniae, while Dokhwal, Nadosongipul, and Sumbadi against S. parauberis and S. iniae, and Mulchamnamu, Seoeonamu, and Seipijilpul against V. ordalii.

Biochemical, serological and molecular properties of a group of 14 Vibrio ordalii strains isolated from cultured Atlantic salmon Salmo salar in Chile in recent years were studied. The charac- teristics of isolates were compared with the type strain V. ordalii ATCC 33509 T. The Chilean V. ordalii represented a biochemically homogenous group; however, some minor differences with the type strain were observed. The serological relationships among isolates, as well as the study of their anti- genic determinant (LPS) revealed a strong reaction with antisera raised against Atlantic salmon strains and the antiserum raised against Listonella anguillarum serotype O2. However, LPS elec- trophoretic patterns were completely different from the V. ordalii type strain, regardless of the serum employed, suggesting the possibility that the Chilean strains constitute a new serological subgroup within this bacterial species. Genetic analyses by PFGE, RAPD, REP-PCR and ERIC-PCR demon- strated that all V. ordalii strains were genetically homogenous, displaying similar DNA patterns, regardless of the techniques used. Moreover, the analysis of DNA banding patterns generated by ERIC-PCR and REP-PCR also clearly separated the type strain from the Chilean strains. This is the first report of characterization of V. ordalii strains from the Southeastern Pacific area, the results of which should facilitate the development of vaccines for protecting cultured Atlantic salmon against vibriosis in this area.

The objective of this research was to know the effectiveness of polyvalen Vibrio vaccine to control vibriosis in humpback grouper (Cromileptes altivelis) . The effectiveness of vaccine was evaluated by the survival rate (SR), relative percent survival (RPS), mean time to death (MTD) as well as growth rate of vaccinated fish. This research consisted of 4 treatments (control, injection, immersion, and oral vaccinations) in quadruplicates. Injection vaccination was conducted by intraperitoneal injection of polyvalen vaccine at 10 7 cells/fish. Immersion vaccination was done by immersing the fishes at 10 7 cells/ml for 30 minutes. Oral administration of vaccine was also carried out at 10 7 cells/fish. One week after the first vaccination, second vaccination (booster) was carried out at the same dosage and by the same administration. One week after the second vaccination, fishes were challenged with 3.16x10 4 cells/fish of Vibrio ordalii 3J by intraperitoneal injection, and reared for 20 days post infection. Results indicated that polyvalen Vibrio vaccine increased SR (P

Vibrio ordalii, the causative agent of atypical vibriosis, is a Gram-negative, motile, rod-shaped bacterium that severely affects the salmonid aquaculture industry. V. ordalii has been biochemically, antigenically and genetically characterized. However, studies on the survival behaviour of this bacterium in aquatic environments are scarce, and there is no information regarding its disease transmission and infectious abilities outside of the fish host or regarding water as a possible reservoir. The present study investigated the survival behaviour of V. ordalii Vo-LM-06 and Vo-LM-18 in sterile and non-sterile seawater microcosms. After a year in sterile seawater without nutrients, 1% of both V. ordalii strains survived (~10(3) colony-forming units ml(-1)), and long-term maintenance did not affect bacterial biochemical or genetic properties. Additionally, V. ordalii maintained for 60 d in sterile seawater remained infective in rainbow trout Oncorhynchus mykiss. However, after 2 d of natural seawater exposure, this bacterium became non-culturable, indicating that autochthonous microbiota may play an important role in survival. Recuperation assays that added fresh medium to non-sterile microcosms did not favour V. ordalii recovery on solid media. Our results contribute towards a better understanding of V. ordalii survival behaviour in seawater ecosystems.

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 × 108 cells g−1 of feed, JB-1 led to a reduction in mortalities to 0–13% after challenge with a range of bacterial pathogens compared to 80–100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0–16% following the challenge compared to 80–100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.

Rainbow trout (Oncorhynchus mykiss) head kidney phagocytes precultured with a synthetic cytidine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN) displayed significantly higher phagocytic activity against Vibrio ordalii than phagocytes precultured with non-CpG ODN. However, head kidney phagocytes precultured with CpG ODN did not show enhanced phagocytic activity against polystyrene particles.

The bacterium Vibrio ordalii is best known as the causative agent of vibriosis outbreaks in fish and thus recognized for generating serious production losses in aquaculture systems. Here we report for the first time on the isolation and the genome sequencing of phage vB_VorS-PVo5, infectious to Vibrio ordalii ATCC 33509. The features as well as the complete genome sequence and annotation of the Vibrio phage are described; vB_VorS-PVo5 consists of a lineal double stranded DNA totaling ~ 80.6 Kb in length. Considering its ability to lyse Vibrio ordalii ATCC 33509, the phage is likely to gain importance in future aquaculture applications by controlling the pathogen and as such replacing antibiotics as the treatment of choice.

The rapid haemostasis of fish prevents bleeding or infection that could be caused by physical properties of the aquatic environment. Additionally, the innate immune system is the first line of defence against infection and is responsible for the recognition of pathogen-associated molecular patterns, which are important for the activation of acquired immune responses. Coagulation factor II (CFII) is an important factor in the coagulation system and is involved in recognition and interaction with various bacterial and extracellular proteins. In this study, we identified and characterised the gene encoding CFII in rock bream (Oplegnathus fasciatus) (RbCFII) and analysed its expression in various tissues after a pathogen challenge. The full-length RbCFII cDNA (2079 bp) contained an open reading frame of 1854 bp encoding 617 amino acids. Alignment analysis revealed that a gamma-carboxyglutamic acid-rich domain, two kringle domains, and a trypsin-like serine protease domain of the deduced protein were well conserved. RbCFII was ubiquitously expressed in all tissues examined but, predominantly detected in the liver and skin. RbCFII expression was dramatically up-regulated in the kidney, spleen and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. The recombinant protein RbCFII (rRbCFII) produced using an Escherichia coli expression system was able to bind all examined bacteria. Interestingly, rRbCFII has agglutination activities towards E. coli and E. tarda, while no agglutination was shown toward Vibrio ordalii and S. iniae. These findings indicate that rRbCFII performs an immunological function in the immune response, and might be involved in innate immunity as well as blood coagulation.

Sablefish Anoplopoma fimbria are a prized seafood species due to their high oil content and white flaky flesh. Raising these species in culture can help to provide an important source of protein for humans and relief to declining wild fish populations. Understanding the environmental factors that influence the production of Sablefish is important for successful culturing. The significance of host-pathogen interactions in Sablefish culture and the resulting environmental implications are unknown. Pathogens could potentially cause losses of cultured Sablefish stocks due to disease, while Sablefish cultured in net pens may also serve as reservoirs for pathogens and potentially transmit disease to wild fish species. In this initial study, the susceptibility of juvenile Sablefish to three bacterial pathogens from the family Vibrionaceae was examined. Listonella anguillarum, Vibrio ordalii, and V. splendidus can pose serious economic threats to cultured fish and shellfish. Groups of juvenile Sablefish were exposed to five concentrations of each of the pathogens. Sablefish were susceptible to L. anguillarum, but were resistant to V. ordalii and V. splendidus at exposure concentrations of ≤ 1.32 × 10⁷ CFU/mL and ≤ 3.57 × 10⁶ CFU/mL, respectively. The greatest L. anguillarum concentration examined (8.7 × 10⁶ CFU/mL) resulted in 24% mortality in juvenile Sablefish. A 24% loss of Sablefish stock could significantly influence an aquaculture program. As determined by multiple logistic regression, the survival of Sablefish to L. anguillarum exposure was significantly affected by their body mass, and larger fish had a greater probability of survival. Aquaculture operations could employ various strategies to minimize the loss of juvenile Sablefish by accounting for their size and known susceptibilities to pathogens.

In this work, 51 V. anguillarum isolates (42/51 of O1 serotype, 9/51 of O2) obtained from cultivated marine fish by Avsever and Un (2015) were used. These isolates were confirmed with universal primers specific to amiB genes. They were also detected successfully at a rate of 100% with a new primer couple designed according to 16S ribosomal RNA sequence. The new primers were found to have a high sensitivity and specificity but were liable to give a cross-reaction with Vibrio ordalii. The IJA appears exclusively as a peer-reviewed on-line open-access journal at http://www.siamb.org.il/. To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden.

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum has been particularly devastating in the marine culture of salmonid fish. The correlation between serotype and virulence may reflect the ability of the bacterial surface antigens to interact with the host tissues. The ferric-anguibactin receptor FatA is an 86-kDa protein that is essential for anguibactin transport. The FatA amino acid sequence is similar to other receptors involved in iron transport, e.g., FhuA and FepA of Escherichia coli, and a TonB box can be identified at its amino-terminal end. V. ordalii causes vibriosis in wild and cultured marine salmonids in the Pacific Northwest of the United States and Japan. The determination and analysis of the complete nucleotide sequence could provide information about the role of pMJ101 in the pathobiology of this fish pathogen. Juvenile salmon exposed to V. ordalii by parenteral challenge developed a systemic infection, and the bacterium was recovered from liver, kidneys, spleen, and blood immediately after the infection. However, the number of bacteria in the liver declined after 1 h and then increased 22 h after infection; bacterial numbers were high in all the organs, and 100% mortality occurred 6 days after infection. The latter observations demonstrate that artificial infection of juvenile salmon is a valid experimental model to study the mechanisms and the bacterial virulence factors involved in the pathogenesis of the infections caused by V. ordalii.

In order to clarify the effect of a prolonged exposure period on vaccine effectiveness, juvenile rainbow trout were immersed in 100, 1, 000 and 10, 000 times dilutions of a commercial vibrio bacterin for 3 min (brief immersion, BI) and for 24 h (prolonged immersion, PI). Two weeks and 2, 4 and 6 mo after the vaccination, fish were challenged by waterborne exposure to Vibrio ordalii. Prolonged exposure to dilute bacterin resulted in better protection than the standard BI method. Two weeks after vaccination, the cumulative mortality was 0% in the group vaccinated by PI with the 100, 1, 000 and 10, 000 times diluted vaccine. In contrast, mortality was 58 and 77% in the groups vaccinated by BI with the 100 and 10, 000 times diluted vaccine respectively, and was 100% in the unimmunized control group. Furthermore, 6 mo after vaccination, the mortality was 20% (100 times dilution) to 77% (10, 000 times dilution) in the PI group, while it was 64% (100 times dilution) to 93% (10, 000 times dilution) in the BI group. Mortality in the corresponding control group was 97%. These results suggest that prolonged exposure period improves vaccine effectiveness.

Many species of pathogenic bacteria have been reported from marine fishes in Japan : Aeromonas salmonicida (atypical), clamydia like organisms (epitheliocystis organisms), Edwardsiella tarda, Flexibacter maritimus, Lactococcus garvieae (syn. Enterococcus seriolicida), Mycobacterium sp., Nocardia seriolae (syn. N. kampachi), Photobacterium damsela subsp. piscicida (syn. Pasteurella piscicida), Pseudomonas anguilliseptica, Pseudomonas putida, Renibacterium salmoninarum (in maricultured salmonids), Staphylococcus epidermidis, Streptococcus equisimilis, Streptococcus iniae, Vibrio alginolyticus, Listonella anguillarum (syn. Vibrio anguillarum), Vibrio ichthyoenteri, Vibrio ordalii, Vibrio trachuri and some other minor or unknown species. These bacteria usually give damages to some limited fish species, however, most of them have been found to be distributed in other cultured and wild fish species which will be possible hosts or carriers of pathogens. Drug resistance, deterioration of water around farms, and spread of pathogens by transporting seedling fish are the major problems for the control of disease in Japanese mariculture.

El objetivo principal de este estudio es estandarizar las metodologías de ensayos de susceptibilidad a agentes antimicrobianos de especies bacterianas patógenas de salmónidos presentes en la salmonicultura chilena. Unpublished

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 (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum, and for serotyping of these pathogenic vibrios.

Thirty strains of V. anguillarum were tested for the production of inhibitory substances against closely-related bacteria using the deferred antagonism test. Only one strain, Vibrio anguillarum VL4355, inhibited strains of V. ordalii and this effect was blocked by the addition of iron salts to the culture medium. Siderophore production was investigated for this strain. Results from bioassays suggested that strain VL4355 produced a siderophore related to anguibactin, the plasmid-encoded phenolate siderophore produced by V. anguillarum strain 775. However, when plasmid DNA was compared for strains 775 and VL4355 the BamHI-generated restriction profiles were different, although hybridization experiments indicated some homology. Using the chrome-azurol sulphate assay to measure siderophore production, strain VL4355 yielded significantly higher values than other V. anguillarum strains. Amberlite XAD-2 was used to produce concentrated siderophore preparations from strains VL4355 and 775. Both preparations were inhibitory to the growth of strains of V. ordalii, but not V. anguillarum, as were solutions of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The difference in sensitivity to iron-limiting conditions for V. ordalii and V. anguillarum, coupled with the inability of V. ordalii to utilize ferric-anguibactin, could reflect different mechanisms of iron uptake for these two organisms.

Eighty regional strains of Vibrio isolated from the seasonally cold waters of coastal Newfoundland, and a number of Vibrio reference cultures, were studied. The regional strains had been isolated from the brown macroalga Alaria esculenta and the giant scallop Placopecten magellanicus and were known to grow at 4 °C. The strains were grouped according to their arginine-dihydrolase reactions and examined by numerical analysis. According to phenotypic properties the arginine-dihydrolase positive strains closely resembled Vibrio splendidus biovar I. Most clusters of the arginine-dihydrolase negative strains appeared to be unique but the closest phenotypic resemblance among some strains was with Vibrio ordalii. Some strains were examined using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique for fingerprinting and it was shown that the regional strains were significantly different from either V. splendidus biovar I or V. ordalii. Generally, the strains from seaweed clustered separately from those that were from scallops. Strains in some clusters, especially those from the seaweed, were able to utilize most of the compounds that were tested as sole sources of carbon and energy.Key words: numerical taxonomy, marine bacteria, Vibrio, psychrotrophs, RAPD-PCR.

The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far. The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants. Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions. However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein. Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein. The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E. coli strain. Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M. Couturier et al. (Microbiol. Rev. 52, 375-395, 1988).

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437T and ATCC 68554 ( = strain 775) was Vibrio ordalii LMG 13544T, with more than 99.5 % 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544T, with 95 % AAI, 98 % MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77 % similarity in AAI, 85 % in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67 % DNA–DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges–Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

The efficacy of the disinfectant Duplalim®, a synergistic blend of superquats and glutaraldehyde, was analysedin vitroagainst 4 fish pathogens. All concentrations tested (1:200 to 1:400 dilutions) were efficacious on killingVibrio ordaliiandVibrio anguillarumin seawater after 30 s, being the level of reduction equal to 1.8 x 106CFU/ml. Higher concentration of Duplalim®(1:50 dilutions) and time of exposure (at least 5 min) is needed to kill completelyFrancisellasp, an intracellular freshwater pathogen. When Infectious Pancreatic Necrosis Virus (IPNV) was treated with 1:400 disinfectant dilution, this concentration had a significant effect after 2 minutes, regardless of the IPNV titres employed (concentration greater than 107.6TCID50/ml). Duplalim®was tested in large scale againstVibrionaceaemembers. In comparison to the controls (without the disinfectant), 1:400 dilutions of Duplalim®totally killedV. ordaliiandV. anguillarumin seawater as well as on the surface of the fishing net (used in the cages of cultured salmon) after 15 min. Cultivable bacteria remained constant in the buckets without the disinfectant (1.4 ± 0.3 × 106CFU/ml), regardless of the period sampled. In the case of the adherence on the fishing net, bacteria not exposed to the disinfectant were detected at a concentration of 7.6 ± 3.2 × 105CFU/ml. These data indicate that the use of Duplalim®againstV. ordalii,V. anguillarumand IPNV is effective in low concentration and short time of exposure (15 min at a concentration of 1:400 dilutions), while the intracellular pathogen requires higher concentration. &nbsp, En el presente trabajo se evaluó la eficaciain vitrodel desinfectante Duplalim®, una combinación sinérgica de glutaraldehído y sales de amonio cuaternario de cuarta generación, contra 4 patógenos de peces prevalentes de la salmonicultura chilena. Los resultados muestran que todas las concentraciones ensayadas (diluciones entre 1:200 a 1:400) fueron eficaces sobre los aislados deVibrio ordaliiyVibrio anguillarumpost-30 s de exposición, detectando niveles de reducción igual a 1.8 x 106UFC/ml. Concentraciones superiores de Duplalim®(dilución 1:50) y un tiempo de exposición no menor a 5 min. Fueron necesarios para eliminar completamente al patógeno intracelularFrancisellasp. Cuando el desinfectante fue ensayado contra el Virus de la necrosis Pancreática infecciosa (IPNV), se detectó que la dilución 1:400 tiene un efecto significativo después de 2 minutos sin importar los títulos de IPNV testeados (mayor concentración evaluada 107.6TCID50/ml). Duplalim®se evaluó en condiciones masivas contra los miembros de la familiaVibrionaceae. En comparación a los controles (sin adición desinfectante), la dilución 1:400 de Duplalim®eliminó completamenteV. ordaliiyV. anguillarumdespués de 15 minutos de tratamiento, tanto en el agua de cultivo como en la superficie de mallas usadas en el cultivo del salmón. Así, el análisis microbiológico del agua de los controles mostró concentraciones de 1.4 ± 0.3 × 106UFC/ml, mientras en el caso de las mallas 7.6 ± 3.2 × 105UFC/ml1. En resumen, los antecedentes obtenidos indican que el uso del desinfectante Duplalim®es efectivo contraV. ordalii,V. anguillarumy IPNV en bajas concentraciones y cortos periodos de exposición (dilución 1:400 por 15 min.), mientras que para el patógeno intracelular se requiere una concentración mayor. &nbsp

Polyclonal rabbit antisera raised against Vibrio ordalii and serotype 02 strains of Vibrio anguillarum showed extensive cross-reactivity with lipopolysaccharide from these bacterial pathogens of fish when tested in western immunoblot analysis. Results with absorbed polyclonal antisera indicated that lipopolysaccharide molecules from these strains had both common and strain-specific antigenic determinants, which allowed the antisera to be used to differentiate between V. ordalii and serotype 02 strains of V. anguillarum. Unlike rabbits, the immune response in rainbow trout to serotype 02 common antigenic epitopes was dependent on the source of the immunizing lipopolysaccharide antigens. Serum from fish immunized with V. ordalii antigens reacted more extensively with serotype 02 common antigens. In contrast, fish anti-V. anguillarum 02 serum did not interact with O antigens from the V. ordalii strains. Lipopolysaccharide from V. anguillarum serotype 02 and 02a strains showed identical antibody binding properties when interacted with rabbit or fish antiserum to either V. anguillarum 02 or V. ordalii. Lipopolysaccharide from V. anguillarum 02b strains did not interact with the tested rabbit or fish polyclonal sera. The results from this study suggest that fish and rabbits recognise different antigenic determinants in lipopolysaccharide from V. ordalii and serotpye 02 V. anguillarum strains; that V. ordalii and serotype 02 strains of V. anguillarum should be regarded as distinct serotype 02 subgroups based on the strain-specific antigenic determinants; and finally that the serological classification of V. anguillarum serotype 02b strains should be reexamined.Key words: antigenic heterogeneity, lipopolysaccharides, fish immune response.not available

Vaccination of fish for the prevention of specific bacterial diseases afffecting commercially reared fish species has had a significant impact on this industry. Almost all of the vaccines available at this time are bacterins or formalin-inactivated whole cell suspensions, some with adjuvants. The first vaccines to be successfully commercialized were those against Vibrio anguillarum, Vibrio ordalii , and Yersinia ruckeri in the late 1970s. Developed initially for the salmonid industry, these bacterins are now routinely used worldwide on many species of fish. Though in some areas salmon farming has flourished without the use of these vaccines, in most areas they have been essential to the economic viability of aquaculture operations. Vaccines against Vibrio salmonicida , a pathogen of salmonids, Aeromonas salmonicida , a pathogen of salmonids and carp, and Edwardsiella ictaluri , a pathogen of channel catfish have also been commercialized and are in widespread use. A number of other bacterins have been the subject of research and some of them may eventually be available. Though a bacterin against Vibrio parahaemolyticus , a pathogen affecting species of fish reared in warmwater has been successfully tested, as have bacterins against Aeromonas hydrophila and Edwardsiella tarda , the serologic heterogeneity of these groups of organisms make it unlikely that widely utilizable vaccines will be available in the near future. Those pathogens that appear to be serologically more homogeneous, including Flexibacter columnaris, Pasteurella piscicida and Streptococcus species affecting fish, will likely end up in commercially available bacterins in the not too distant future. The use of a new generation of adjuvants in conjunction with automated injection methods could result in vaccines that will protect against diseases that conventional methods may not be successful against, such as bacterial kidney disease (BKD) caused by Renibacterium salmoninarum .

The phenotypical, serological, and molecular identification of Vibrio ordalii isolates recovered from diseased gilthead sea bream (Sparus aurata) in the Mediterranean area were investigated. Most isolates had similar phenotypic characteristics, but some showed small differentiation in biochemical analysis. Both slide agglutination and enzyme linked immunosorbent assay (ELISA) showed that the isolates were antigenically homogeneous and no different serotypes were detected. SDS-PAGE analysis showed that the structure of lipopolysaccharide (LPS) was heterogeneous, that outer membrane proteins (OMP) display homogeneous grouping protein banding patterns, and that the LPS profile of the isolates had different banding patterns. However, random amplified polymorphic DNA (RAPD) analysis revealed genetic variation (up to 90.4%) among isolates of the same species.

We examined genetic variation in mortality and mean times to death for three British Columbia populations of chinook salmon Oncorhynchus tshawytscha, subgroups of which were challenged with one of three pathogens: Vibrio anguillarum, which causes vibriosis; Aeromonas salmonicida, which causes furunculosis; or Renibacterium salmoninarum, which causes bacterial kidney disease. In each population, 10 males were mated to 20 females in a nested breeding design, the progeny were divided into groups, and each group was challenged with a pathogen. Heritability estimates (sire component, binary character) of mortality rate were 0.00 for vibriosis, 0.00–0.34 for furunculosis, and 0.00–0.38 for bacterial kidney disease. Estimates of heritabilty for time to death were 0.00 for vibriosis, 0.00–0.02 for furunculosis, and 0.06–0.50 for bacterial kidney disease. There was little correlation among mortality rates for the different pathogens for the families examined in each population. Kitimat River chinook salmo...

We examined variation in mortality and mean time to death for four British Columbia populations of pink salmon Oncorhynchus gorbuscha, subgroups of which were challenged with Vibrio anguillarum, V. ordalii, or Aeromonas salmonicida. Two hundred fish in each population were challenged with one of the three pathogens. Mortality in the four populations ranged from 84 to 91% after challenge with V. anguillarum, from 13 to 26% after challenge with V. ordalii, and from 51 to 63% after challenge with A. salmonicida. A significant difference in mortality among populations was observed only in the V. ordalii challenge. No significant differences in time to death were observed.

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 108 cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 106 ml−1 from 3·7 ± 0·8 × 106 ml−1), erythrocytes (1·2 ± 0·1 × 108 ml−1 from 0·8 ± 0·1 × 108 ml−1), protein (23 ± 4·4 mg ml−1 from 16 ± 1·3 mg ml−1), globulin (15·7 ± 0·2 mg ml−1 from 9·9 ± 0·1 mg ml−1) and albumin (7·3 ± 0·2 mg ml−1 from 6·1 ± 0·1 mg ml−1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml−1 from 40 ± 8·0 units ml−1), lysozyme (920 ± 128·8 units ml−1 from 760 ± 115·3 units ml−1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.

Isolates of Vibrio ordalii were recovered from salmon (Oncorhynchus tshawytscha and Oncorhynchus nerka) cultured in New Zealand. The fish suffered from clinical vibriosis, from seven outbreaks in five geographically distinct areas. Affected fish showed gross signs and histopathology similar to those reported from North America. The presence of a plasmid, designated pVO1, approximately 30 kilobases (kb) in size, was seen in all V. ordalii strains examined This plasmid was identical in size and restriction endonuclease cleavage analysis to a plasmid (pMJ101) previously found in V. ordalii strains elsewhere. After togging pVO1 with the transposon Tn1 from the R plasmid RP4, attempts to isolate plasmid‐less derivatives using a variety of curing agents were unsuccessful. All of the New Zealand isolates possessed identical chromosomal DNA cleavage patterns. These patterns were only slightly different from that of the type strain but substantially different from that of the Vibrio anguillarum type strain.