Vibrio anguillarum

Proteomic analyses techniques are considered strong tools for identifying and quantifying the protein contents in different organisms, organs and secretions. In fish biotechnology, the proteomic analyses have been used for wide range of applications such as identification of immune related proteins during infections and stresses. The proteomic approach has a significant role in understanding pathogen surviving strategies, host defence responses and subsequently, the fish pathogen interactions. Proteomic analyses were employed to highlight the virulence related proteins secreted by the pathogens to invade the fish host's defence barriers and to monitor the kinetics of protein contents of different fish organs in response to infections. The immune related proteins of fish and the virulence related proteins of pathogens are up or down regulated according to their functions in defence or pathogenesis. Therefore, the proteomic analyses are useful in understanding the virulence mechanisms of microorganisms and the fish pathogen interactions thereby supporting the development of new effective therapies. In this review, we focus and summarise the recent proteomic profiling studies exploring pathogen virulence activities and fish immune responses to stressors and infections.

Vibrios were isolated from red tides caused by Mesodinium rubrum and also throughout the year in the Ria de Pontevedra, Spain. The isolates were grouped into 14 phena by numerical taxonomy. Strains associated with red tides were restricted to four phena: phena I and II were Vibnio alginolyticus, and phena III and IV were Vibrio tubiashii and Vibrio anguillarum, respectively. V. anguilarum-like strains (phena V through XI) predominated throughout the year outside the red tide areas. Cytotoxicity assays conducted in different poikilothermic and homoiothermic cell lines showed that cytotoxin production was not necessarily associated with the species selected during the red tides. Mesodinium rubrum is one of the most interesting organ- isms that causes red tides all over the world (1, 8, 15, 17, 19, 21, 23, 25) because it has characteristics of both photoplank- ton and zooplankton. Blooms produced by this fast-swim- ming primary producer are commonly observed in near- shore areas, in regions of ocean upwelling, and in fjords and sheltered bays (18). Galician rias (Northwest Spain), a very important environment for marine aquaculture of fish and shellfish, are subjected to continuous studies of plankton, blooms of M. rubrum being observed with an annual fre- quency (9, 14, 19). Species of the genus Vibrio are often found in estuarine and seawater environments and have an important role in the decomposition of organic matter, especially of zooplankton (13). Marine vibrios include a great diversity of pathogenic species such as V. parahaemolyticus, V. alginolyticus, and V. cholerae 01 and non-O1, causing diseases in homoiother- mic animals (6, 10); V. anguillarum, V. tubiashii, V. car- chariae, and V. ordalii, pathogens for fish and shellfish (5, 28); and V. vulnificus and V. damsela, infecting both poiki- lothermic and homoiothermic animals (3, 11, 26). The patho- genicity for fish of classic environmental species like V. splendidus has been recently reported (B.

Introduction: In the present study, some of the commercial fish farms located in the Black Sea region of Turkey, were screened for bacteria between 2012 and 2014. Material and Methods: The bacterial agents isolated from fish were identified by classical biochemical tests and the rapid diagnostic tests (API 20 E and API 20 Strep). All strains were further identified by sequencing of the 16S rRNA genes. The strains were also investigated for resistance to different antimicrobials by the disc diffusion method. Antibiotic resistance genes, including tetracycline (B), β-lactam (ampC, blaTEM, blaPSE ), florfenicol (floR), erythromycine (ereA, ereB), sulphonamide (sulI, sulII), and trimethoprim (dhfr1) genes, were determined by the PCR method. Results: Vibrio anguillarum, Vibrio fluvialis, Photobacterium damselae subsp. piscicida, Pseudomonas luteola, Lactococcus garvieae, Streptococcus iniae, Aeromonas hydrophila, and Yersinia ruckeri were isolated from marine and freshwater cultured fish...

The purpose of this study was to examine the cumulative effects of exposure to a pathogenic bacteria and municipal effluent in the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations of an ozone-treated effluent at 15°C for 7days. A sub-group of mussels was inoculated with Vibrio anguillarum and exposed to the same conditions as above. After the exposure period, mussels were collected to assess hemocyte count and viability, immunocompetence (phagocytosis and nitrite production), oxidative stress/inflammation (cyclooxygenase and lipid peroxidation) and oxygen radical/xenobiotic scavenging activity (metallothioniens, glutathione S-transferase). The results showed that mussels exposed to municipal effluent had increased hemocyte counts, phagocytosis, nitrites, lipid peroxidation and metallothioneins. In the inoculated mussels, the same responses were observed, in addition to cyclooxygenase and glutathione S-transferase activities. Multivariate analy...

The dietary selenium (Se) requirement of yellowtail kingfish (Seriola lalandi) in normal and infected conditions was investigated. The fish were fed one of five experimental diets; a control un-supplemented diet (3.35 mg/kg Se) or diets supplemented with Se to provide 4.86, 5.38, 5.85 or 6.38 mg/kg Se. After feeding for 6 weeks, the fish were challenged by Vibrio anguillarum immersion and then observed for 4 weeks. Supplementation of Se had no effect on feed intake, feed conversion ratio and survival over 6 weeks of feeding; however, it significantly increased growth and Se content in muscle tissues. The optimal Se level for maximal growth of yellowtail kingfish estimated by second order regression was 5.56 mg/kg. Following the bacterial challenge, the immune-stimulating effects of Se were demonstrated in lysozyme and bactericidal activities, and there was a corresponding increase in survival and antibody response by supplementation of Se at ≥2 mg/kg (measured Se of ≥5.38 mg/kg). Under normal and infectious conditions, antioxidant capacity of fish measured as glutathione peroxidase activity increased by supplementation of Se. During post-challenge period, haematocrits were higher in the fish fed Se supplemented diets than the fish fed the control diet, while more macrophage aggregates were seen in the control group than in the others. Furthermore, there was evidence of myopathy in fish fed the diet without Se supplementation. Therefore, the results indicated that the optimal dietary Se requirement of yellowtail kingfish is 5.56 mg/kg.

) on immune competence of yellowtail kingfish, Seriola lalandi, were investigated. The fish were fed one of three experimental diets including a control diet without Se supplementation and two diets supplemented with Se from Seyeast (Selplex®) at 2 and 4 mg/kg. After feeding for 6 wk, the fish were challenged by injecting Vibrio anguillarum and observed for 2 wk. Dietary Se had no effect on feed intake, feed conversion ratio and survival over the course of 6-wk feeding, however, it significantly increased weight gain and Se content in muscle. Following the bacterial infection, the immune-stimulating effects of Se were observed in antibody, lysozyme and bactericidal responses, and there was a corresponding increase in survival and haematocrit by Se. Under infectious condition, antioxidant capacity of fish as measured in term of resistance of red blood cells to peroxidation and glutathione peroxidase activity also increased by supplementation of Se. Liver necrosis and kidney melanomacrophages were only seen in surviving fish fed the control diet after the challenge. Furthermore, there was evidence of myopathy in fish fed the diet without Se supplementation. This study suggests that Se, supplemented at 2 or 4 mg/kg, can improve growth and health of yellowtail kingfish.

Thirty-one strains of Vibrio anguillarum serogroup O1 isolated in Italy and Greece from dead fish were subjected to rRNA gene restriction pattern analysis. With HindlII as the restriction enzyme, two different ribotypes were detected, of which one profile was dominant. One profile was found in all strains except one isolated from sea bass, sea bream, and mullet, while the second profile was found in all three strains isolated from rainbow trout and in one strain from a sea bass. With EcoRI only one profile was found.

The use of natural immunostimulants is considered the most promising alternative to promote fish health, productive performance and quality, increasing the aquaculture profitability, sustainability and social acceptance. The purpose of this study was to evaluate the effect of the integration of a potential probiotic strain, Halobacterium salinarum, belonging to the Archaea domain, in the formulated diets of farmed gilthead seabream (Sparus aurata L.) in terms of growth performances and immunity responses. The experiment was set up to test two different levels of inclusion of the bacteria in the diet: 0.05% (D1) and 0.1% (D2). The effects on fish growth performances; humoral (peroxidase, protease, antiprotease and IgM levels) and cellular immunity parameters (phagocytosis, respiratory burst and myeloperoxidase), along with bactericidal activity, were evaluated after 15 and 30 days of experimental feeding. The obtained results showed that the inclusion of H. salinarum at the highest c...

A Pseudomonas sp. was cultured which was associated with the Japanese seaweed Diginea sp. Crude extracts prepared from this bacterial culture were found to inhibit the growth of other marine bacterial strains. From this bacterial culture, two new peptides cyclo-[phenylalanyl-prolyl-leucylprolyl] (3) and cyclo-[isoleucyl-prolyl-leucyl-alanyl] (4) have been isolated together with two known peptides (1) and (2). The crude extract from a culture of Pseudoalteromonas sp. associated with the Thai sponge Halisarca ectofibrosa was found to inhibit the growth of Bacillus subtilis and Vibrio anguillarum. Isolation studies yielded a fraction containing two peptides that were identified as cyclo-[phenylalanyl-leucyl] 2 (5) and cyclo-[leucylisoleucyl] 2 (6) by means of LC-MS and 2D NMR data. Absolute stereochemistry was confirmed by the synthesis of cyclo-[L-phenylalanyl-L-leucyl] 2 . Peptides (1)e(3) were also isolated from this bacterial strain. None of the individual peptides isolated in this study showed antibiotic activity.

Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, ) and a marine fish (gilthead seabream, ) to bacterial immersion ( bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress- and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was m...

Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, ) and a marine fish (gilthead seabream, ) to bacterial immersion ( bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress- and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was m...

Chemokines are cytokines that can promote the activation and migration of immune cells, and increase the recognition of antigen by antigen-presenting cells (APC). Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of fl ounder after immunization. To explore the improvement of chemokines on the effi ciency of OmpK vaccine, two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the fl ounder were investigated by reverse transcription polymerase chain reaction (RT-PCR), indirect immunofl uorescent assay (IFA), H&E staining, fl ow cytometry (FCM), and quantifi cational real-time polymerase chain reaction (qRT-PCR). pBudCE4.1 plasmid as an expression vector, bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene (p-OmpK-CCL4), or Ompk gene and CC-motif ligand 19 gene (p-OmpK-CCL19) were successfully constructed. The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/ CCL19 proteins of fl ounder both in vitro and in vivo. After immunization, a large number of leucocytes in muscle were recruited at the injection site in treatment groups. The constructed vaccines induced signifi cant increases in CD4-1 + and CD4-2 + T lymphocytes, and sIgM + B lymphocytes in peripheral blood, spleen, and head kidney. The percentage of T lymphocytes peaked on the 14 th post-vaccination day whereas that of B lymphocytes peaked in the 6 th post-vaccination week. Moreover, the expression profi les of 10 immune-related genes increased in muscles around the injection site, spleen, and head kidney. After the challenge, p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival (RPS) of 74.1% and 63.3%, respectively, higher than p-OmpK alone (40.8%). In conclusion, both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity. CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single‐egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty‐nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake ha...

Armoracia rusticana (AR) was tested for antimicrobial and antioxidants power. The compound demonstrated to inhibit fish pathogens such as Vibrio anguillarum, V. harvey, V. alginolyticus, Aeromonas hydrophila, A. salmonicida, Photobacterium damselae subspecie piscicida, Tenacibaculum marinum and Pseudomonas anguilliseptica,. The total phenolic content and the reducing power resulted higher in the water extract of AR, respect to the hydroalcoolic. In vitro test demonstrated that AR significantly protect cells against death, induced by oxidative stress.

The rainbow trout production increased more than 100% in the last decade and total rainbow trout production was showed as 107.013 according to 2016 statics. According to Federation of European Aquaculture Producer, Turkey was determined as the biggest rainbow trout producer into the European Countries in 2016. Together with high production capacity, a number of outbreaks were reported causing L. anguillarum (Vibriosis). The pathogenesis of L. anguillarum which were experimentally infected in rainbow trout were examined during 15 days in our study. Spreading of agent that was injected with intraperitoneally into tissue and organs were studied by using histopathologic methods. The mortality rate of agent was determined above 70% and deaths were seen in 2-3th days of experiment. In addition of these, liver, spleen, kidney and gills were determined as the most affected organs and tissues. In the present study obtain original pathological findings of L. anguillarum.

Bee venom (BV) is a rich source of secondary metabolites from honeybees (Apis mellifera L.). It contains a variety of bioactive ingredients including peptides, proteins, enzymes, and volatile metabolites. This study investigated the antibacterial effects of the bee venom obtained from honey bees (Apis mellifera L.) against bacterial fish pathogens, such as Lactococcus garvieae (Lg1, Lg2, Lg3), Vibrio anguillarum (Va1, Va2, Va3), Yersinia ruckeri (Yr1, Yr2, Yr3), and Aeromonas hydrophila (Ah1, Ah2, Ah3) and the expression levels on the antibiotic resistance genes hly and fbp (hemolysin and fibronectin-binding prot) of them. It was determined that bee venom had an antibacterial effect against L. garvieae, L. anguillarum, and Y. ruckeri strains, while it had no effect only against Ah3 and Ah2 bacterial strains. As staded by the gene expression of hly (hemolysin) and fbp (fibronectin-binding protein), among the antibiotic resistance genes the effect levels of bee venom on bacterial spec...

Vanchrobactin, a new catecol-type siderophore produced by cells of the fish pathogen Vibrio anguillarum serotype O2, has been isolated from the supernatants of iron-deficient cultures. Its structure was characterized from spectral data and established as N-[N 0 -(2,3-dihydroxybenzoyl)-arginyl]-serine.

Zonula occludens toxin (Zot) is a conserved protein in filamentous vibriophages and has been reported as a putative toxin in Vibrio cholerae. Recently, widespread distribution of zot-encoding prophages was found among marine Vibrio species, including environmental isolates. However, little is known about the dynamics of these prophages beyond V. cholerae. In this study, we characterized and quantified the zot-encoding filamentous phage VAIϕ, spontaneously induced from the fish pathogen V. anguillarum. VAIϕ contained 6117 bp encoding 11 ORFs, including ORF8pVAI, exhibiting 27%–73% amino acid identity to Inovirus Zot-like proteins. A qPCR method revealed an average of four VAIϕ genomes per host genome during host exponential growth phase, and PCR demonstrated dissemination of induced VAIϕ to other V. anguillarum strains through re-integration in non-lysogens. VAIϕ integrated into both chromosomes of V. anguillarum by recombination, causing changes in a putative ORF in the phage genome...

Temperate ϕH20-like phages are repeatedly identified at geographically distinct areas as free phage particles or as prophages of the fish pathogen Vibrio anguillarum. We studied mutants of a lysogenic isolate of V. anguillarum locked in the quorumsensing regulatory modes of low (ΔvanT) and high (ΔvanO) cell densities by in-frame deletion of key regulators of the quorum-sensing pathway. Remarkably, we find that induction of the H20-like prophage is controlled by the quorum-sensing state of the host, with an eightfold increase in phage particles per cell in high-cell-density cultures of the quorum-sensingdeficient ΔvanT mutant. Comparative studies with prophage-free strains show that biofilm formation is promoted at low cell density and that the H20-like prophage stimulates this behavior. In contrast, the high-cell-density state is associated with reduced prophage induction, increased proteolytic activity, and repression of biofilm. The proteolytic activity may dually function to disperse the biofilm and as a quorum-sensing-mediated antiphage strategy. We demonstrate an intertwined regulation of phage-host interactions and biofilm formation, which is orchestrated by host quorum-sensing signaling, suggesting that increased lysogeny at high cell density is not solely a strategy for phages to piggy-back the successful bacterial hosts but is also a host strategy evolved to take control of the lysis-lysogeny switch to promote host fitness.

ABSTRACTSelection for phage resistance is a key driver of bacterial diversity and evolution, and phage-host interactions may therefore have strong influence on the genetic and functional dynamics of bacterial communities. In this study, we found that an important, but so far largely overlooked, determinant of the outcome of phage-bacterial encounters in the fish pathogenVibrio anguillarumis bacterial cell-cell communication, known as quorum sensing. Specifically,V. anguillarumPF430-3 cells locked in the low-cell-density state (ΔvanTmutant) express high levels of the phage receptor OmpK, resulting in a high susceptibility to phage KVP40, but achieve protection from infection by enhanced biofilm formation. By contrast, cells locked in the high-cell-density state (ΔvanΟmutant) are almost completely unsusceptible due to quorum-sensing-mediated downregulation of OmpK expression. The phenotypes of the two quorum-sensing mutant strains are accurately reflected in the behavior of wild-typeV...

Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs), genetic diversifications were located in three variable regions (VR1, VR2 and VR3) in six of the phage genomes. Identification of specific genes, such as N6-adenine methyltransferase and lambda like repressor, as well as the presence of a tRNA(Arg), suggested a both mutualistic and parasitic interaction between phages and hosts. During short term phage exposure experiments, 28% of a V. anguillarum host population was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V...

Manganese (Mn) is essential for normal growth, reproduction and prevention of skeletal abnormalities in terrestrial animals 1 and fish. Manganese acts through its ability to sustain proper enzyme activity either as a metal cofactor for a number of enzymes which form metalenzyme complexes (i.e. glycosyl transferases and glycosaminoglycans), or as an integral part of certain metalloenzymes. The latter include arginase, pyruvate carboxylase, avimanganin and Mn-containing superoxide dismutase (SOD), Mn-SOD. 1 There is also some evidence that Mn is involved in immunological functions. Signs of Mn deficiency in fish have been reported for rainbow trout Oncorhynchus mykiss, 5-7 carp Cyprinus carpio, and tilapia Tilapia mossambica. 9 Manganese deficiency was associated with poor growth, depressed feed intake, skeletal abnormalities (dwarfism), eye lens cataracts, increased mortality, and low tissue Mn. The activities of Cu, Zn-SOD and Mn-SOD both in liver and heart muscle were reduced as a consequence of Mn deficiency in rainbow trout. The quantitative requirement for Mn in fish has been reported to be around 13 mg/kg diet for rainbow trout, 5 12-13 mg/kg diet for carp, 5,10 and 2.4 mg/kg diet for channel catfish. In a previous work, we studied the requirement of Mn supplementation to a fish meal-based diet for Atlantic salmon Salmo salar. 12 Both the concentration and the availability of Mn in different fish meals on the market vary considerably. As a basis for our work with practical recommendations, there is a need for requirement studies based on purified diets correlated to Mn status indicators in the fish. Lall and Bishop demonstrated a qualitative need for Mn supplementation in the diet of Atlantic salmon grown in seawater for optimum growth, food utilization and bone mineralization. 14 However, no

The myeloid differentiation factor 88 (MyD88) is an important adapter protein which links members of the toll-like receptor (TLR) to the downstream components to activate related signaling pathways. In the present study, a MyD88 homolog (FcMyD88) was cloned from penaeid shrimp Fenneropenaeus chinensis. The ORF of FcMyD88 consisted of 1434 bp encoding a polypeptide of 477 amino acids which contains a death domain (DD) and a typical TLR and interleukin-1 receptor (IL-1R)-related (TIR) domain. Homology analysis revealed that the predicted amino acid (aa) sequence of FcMyD88 shared high similarities with a variety of previously reported MyD88s. The time-dependent expression patterns of FcMyD88 in cephalothoraxes of shrimp injected with Vibrio anguillarum (Gram-negative bacteria, G À ), Micrococcus lysodeikticu (Gram-positive bacteria, G þ ) and white syndrome spot virus (WSSV) were analyzed at transcription and protein level by real-time PCR and western blotting, respectively. The expression level of FcMyD88 mRNA was significantly up-regulated at one hour (h), 12 h and 24 h after stimulation with both V. anguillarum and M. lysodeikticu. The expression level of FcMyD88 protein was 2fold up-regulated at 12 h post injection (hpi) of inactivated V. anguillarum while it didn't change after M. lysodeikticu injection during this period. After WSSV injection, the expression level of FcMyD88 mRNA remained relatively constant, while the FcMyD88 protein was significantly up-regulated at 12 and 24 hpi. These results suggested that the MyD88-dependent signaling pathway could be involved in the defense of both bacteria and WSSV infection.

Vibriosis is a bacterial disease in fish caused by the Gram negative bacterium Vibrio anguillarum with severe impact on rainbow trout (Oncorhynchus mykiss) farming. Sustainable control methods should be developed and we here show that marker assisted selective breeding of fish naturally resistant to the disease is feasible. We have validated the use of a single nucleotide polymorphism (SNP) marker SNP AX-89,945,921 (QTL on chromosome 21). The QTL was previously found associated with resistance to vibriosis and described following a genome wide association analysis (GWAS) of trout exposed to the bacterium. For this validation spawners were genotyped by use of the 57 K Axiom®Trout Microarray (Affymetrix) and homozygous male fish carrying the allele with the SNP AX-89,945,921 were then selected and used to fertilize eggs from outbred female trout resulting in fish all carrying the SNP (QTL-fish). Control fish (non-QTL fish) were produced by fertilizing the same batch of eggs by use of ...

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout (Oncorhynchus mykiss). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 107 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish – including survivors – and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The ...

Mariculture in Denmark is based on production of rainbow trout grown two years in fresh water followed by one growth season in sea cages. Although the majority of rainbow trout are vaccinated against the most serious bacterial pathogens -Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Yersinia ruckeri, by the use of commercially available vaccines, disease outbreaks requiring treatment with antibiotics still occur. The present study tested the potential of a new experimental multicomponent vaccine that is based on local bacterial strains, isolated from rainbow trout in Danish waters, and thus custom-designed for Danish rainbow trout mariculture. The vaccination with the multicomponent vaccine resulted in protection against three relevant bacterial diseases (yersiniosis, furunculosis, vibriosis) under experimental conditions. We showed that i.p. injection of the vaccine induced specific antibody responses in trout against the different bacterial antigens and regulated expression of genes encoding SAA,

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin-killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin-killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710-1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60-s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.

This study is the first known observation of biofluorescence in the lumpfish (Cyclopterus lumpus). Individual lumpfish were illuminated with blue excitation lighting for photography with both hyperspectral and filtered multispectral cameras. All photographed juvenile lumpfish (n = 11) exhibited green biofluorescence. Light

In order to clarify the effects of triploidy, phagocytosis, agglutinating antibody titres, complement activity of the serum, chemotaxis and phagocytosis of kidney leucocytes were determined in triploid and diploid, immunised and nonimmunised ayu. Formalin-killed Vibrio anguillarum cells were used as the immunogen. Leucocytes from control triploid ayu showed slightly higher phagocytosis than the diploid ones, but after immunisation no significant differences were detected in agglutinating antibody titres, complement activity and phagocytic activity between triploid and diploid ayu.

Lipopolysaccharides(LPS)were prepared from different serotypes of Vibrio anguillarum, J-O-1,J-O-2 and J-O-3, and Vibrio ordalii. The LPS fractions were obtained by phenol-water extraction followed by Cetavlon treatment and high speed centrifugation. Chemical analysis of V. anguillarum J-O-1, J-O-2 and J-O-3 showed 2.5%,4.8%and 1.3% protein;36.3%,30.0% and 20.0% sugar and 7.1%,11.3% and 6.6%fatty acids. Chemical analysis of V. ordalii showed 17.5%protein,13.8% sugar and 1.4% fatty acids. The monosac charide composition of V. anguillarum J-O-1, J-O-2 and J-O-3 was respcctively:0.1%,0.1%and 0 .1%2-keto-3-deoxyhexose(KDO);3.0%,1.9%and 0.6%pentose;32.6%,21.6%and 12.6% hexose;10.8%,7.6%and 2.8%6-deoxyhexose;4.6%,7.7%and 7.7%heptose;5.1%,6.0%and 10.2%amino sugar. The monosaccharide composition of V. ordalii LPS was:0.0% KDO,1.5% pentose,6.6%hexose,4.0%6-deoxyhexose,0.2%heptose and 3.1%amino sugar. The principal fatty acids of the V. anguillarum lipid A were:myristic acid, palmitic acid and stearic acid. The principal fatty acids constituents V. ordalii lipid A were: lauric acid, palmitic acid, stearic acid and arachidic acid.

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non-transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non-transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and nontransgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth-accelerated coho salmon.

In this study, it was determinated the essential oil of cultivated apple mint, Mentha suaveolens Ehrh. composition and in vitro antibacterial activity of against 11 fi sh pathogen bacteria including Gram-positive (Staphylococcus warneri, Staphylococcus sp., Lactococcus garvieae, Vagococus salmoninarum) and Gram-negative (Aeromonas hydrophila, Aeromonas sobria Aeromonas cavieae, Vibrio anguillarum, Pseudomonas aeroginosa, Yersinia ruckeri, Edwardsiella tarda) by using agar diffusion assay. The main component of M. suaveolence oil was obtained as piperitenone oxide. The essential oil exhibited strong inhibitory activity such as inhibition zone sizes: 30-50mm at 250-1000 µL mL -1 concentrations against V. anguillarum; 16-20mm at 31.25-125 µL mL -1 concentrations against P. aeroginosa; 15-18mm at 500-1000 µL mL -1 concentrations against A. sobria. However, it was found to be moderately effective against E. tarda (8-15 mm), Y. ruckeri (9-12mm), S. warneri (9-10mm), V. salmoninarum (9mm) and Staphylococcus sp. (8-9mm). The essential oil showed weak inhibitory activity against A. cavieae (5-8), A. hydrophila (6-7mm), L. garvieae (5-7mm). Thus, effect of essential oil of M. suaveolens on immune response and disease resistance against Vibrio anguillarum, A. sobria and P. aeroginosa should be investigated in vivo in cultured fi sh species in future studies.

In this work, 51 V. anguillarum isolates (42/51 of O1 serotype, 9/51 of O2) obtained from cultivated marine fish by Avsever and Ün (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.

A Vibrio shaped bacterium has been isolated from nephric tissues of moribund atlantic salmon suffering from the socalled "Hitra disease". Eighteen isolates of this bacterium, from geographically distant fish farms all along the Norwegian coast, were shown to be very similar in biochemical properties. Serotyping and DNA fingerprinting provided additional evidence for similarity between the isolates and that they clearly differ from Vibrio anguillarum and Vibrio ordalii. The particular Vibrio associated with the "Hitra disease" has been designated Vibrio sp. TEO, and is assumed to be involved in the disease development. The bacterium has not been found in any healthy fish, even in farms with outbreaks of "Hitra disease". The Vibrio sp. TEO elicited disease with similar symptoms when injected into healthy fish, and the same bacterium could be reisolated.

Specificities of polyclonal salmon antisera made against the fish pathogens Vibrio salmonicida and Vibrio anguillarum were studied. Using ELISA and Western blot techniques, antisera made against V. salmonicida or V. anguillarum serovar 1 demonstrated high responses against the homologous bacterium or its isolated LPS. In contrast, antisera obtained after immunization with V. anguillarum serovar 2 displayed low antibody titres against homologous antigens. Eleetrophoretic transfer of SDS-PAGE separated V. salmonicida LPS antigen to nitrocellulose strips and subsequent immunostaining with salmon antisera revealed a strong reaction exclusively in the low molecular weight region (<14kD). On the other hand, immunoblots of V. anguiliarum LPS preparations using salmon immunesera raised against this species showed a heterogenous staining pattern ranging from high to medium LPS-size. In addition, most of the salmon antisera made against V. anguillarum serovar 2 also reacted with a low molecular weight LFS antigen band.

Outer membranes and lipopolysaecharides of the marine fish pathogens Vibrio salmonicida and Vibrio anguillarum were isolated. SDS-PAGE profiles of purified LPS preparations from V. salmonicida revealed a broad low moleeular weight band, whereas V. anguillarum LPS profiles demonstrated both a low-moleeular band and several weaker high-moleeular weight bands. Hydrolysis of V. salmonicida and V. angniUarum LPS separating the polysaccharidc chain from the hpid A part and subsequent gcl-chromatography suggests a polysaccharide moleeular weight of ca. 1000 ('rough type' LPS) and ca. 6000 ('smooth type" LPS), respeetively. Western blot of V. salmonicida outer membrane preparations and purified lipopolysaecharides and subsequent immunostaining with mouse monoclonal antibodies was performed. Eleven out of 15 monoelonal antibodies made against V. salmonicida cells reacted with one broad antigen-band in the low moleeular weight region of both outer membrane and LPS profiles, corresponding to the LPS region. The previously reported outer surfaee antigen, VS-Pl from V. salmonicida, was observed to carr>' LPS cpitopes as revealed by binding of monoelonal antibodies to VS-Pl as well as purified LPS preparations. These results strongly suggest that the VS-Pl antigen is a eomplex of both protein and LPS moleeules.

Van Doan H. (2025). Common Carp (Cyprinus carpio) exposure to water-soluble fraction of crude oil (WSF): Evaluation of protective effects of dietary Rosmarinus officinalis leaf on water quality, growth performance, oxidative stress, stress hormones and heat shock proteins, Annals of Animal Science,

Manganese (Mn) is essential for normal growth, reproduction and prevention of skeletal abnormalities in terrestrial animals 1 and fish. Manganese acts through its ability to sustain proper enzyme activity either as a metal cofactor for a number of enzymes which form metalenzyme complexes (i.e. glycosyl transferases and glycosaminoglycans), or as an integral part of certain metalloenzymes. The latter include arginase, pyruvate carboxylase, avimanganin and Mn-containing superoxide dismutase (SOD), Mn-SOD. 1 There is also some evidence that Mn is involved in immunological functions. Signs of Mn deficiency in fish have been reported for rainbow trout Oncorhynchus mykiss, 5-7 carp Cyprinus carpio, and tilapia Tilapia mossambica. 9 Manganese deficiency was associated with poor growth, depressed feed intake, skeletal abnormalities (dwarfism), eye lens cataracts, increased mortality, and low tissue Mn. The activities of Cu, Zn-SOD and Mn-SOD both in liver and heart muscle were reduced as a consequence of Mn deficiency in rainbow trout. The quantitative requirement for Mn in fish has been reported to be around 13 mg/kg diet for rainbow trout, 5 12-13 mg/kg diet for carp, 5,10 and 2.4 mg/kg diet for channel catfish. In a previous work, we studied the requirement of Mn supplementation to a fish meal-based diet for Atlantic salmon Salmo salar. 12 Both the concentration and the availability of Mn in different fish meals on the market vary considerably. As a basis for our work with practical recommendations, there is a need for requirement studies based on purified diets correlated to Mn status indicators in the fish. Lall and Bishop demonstrated a qualitative need for Mn supplementation in the diet of Atlantic salmon grown in seawater for optimum growth, food utilization and bone mineralization. 14 However, no

Background: Microbial diseases cause considerable economic losses in aquaculture and new infection control measures often rely on a better understanding of pathogenicity. However, disease studies performed in fish hosts often require specialist infrastructure (e.g., aquaria), adherence to strict legislation and do not permit high-throughput approaches; these reasons justify the development of alternative hosts. This study aimed to validate the use of larvae of the greater wax moth (Galleria mellonella) to investigate virulence of the important fish pathogen, Vibrio anguillarum. Results: Using 11 wild-type isolates of V. anguillarum, these bacteria killed larvae in a dose-dependent manner and replicated inside the haemolymph, but infected larvae were rescued by antibiotic therapy. Crucially, virulence correlated significantly and positively in larva and Atlantic salmon (Salmo salar) infection models. Challenge studies with mutants knocked out for single virulence determinants confirmed conserved roles in larva and fish infections in some cases (pJM1 plasmid, rtxA), but not all (empA, flaA, flaE). Conclusions: The G. mellonella model is simple, more ethically acceptable than experiments on vertebrates and, crucially, does not necessitate liquid systems, which reduces infrastructure requirements and biohazard risks associated with contaminated water. The G. mellonella model may aid our understanding of microbial pathogens in aquaculture and lead to the timely introduction of new effective remedies for infectious diseases, while adhering to the principles of replacement, reduction and refinement (3Rs) and considerably reducing the number of vertebrates used in such studies.

This study examined culturable skin microbiota that was associated with farmed European seabass (Dicentrarchus labrax). Healthy European seabass were sampled during summer commercial harvest from one conventional fish farm where antibiotics are used, and from another practicing a certified antibiotic-free fish aquaculture. Physicochemical and microbiological analysis of seawater and sediment were performed, as well as determination of culturable bacteria, including Vibrio, from skin swabs of European seabass and seawater and sediment at both farms. Samples were processed for isolation of bacteria and their characterization by molecular and antibiotic susceptibility tests. In both fish farms, most of the bacteria that were identified in the skin belonged to the genera Pseudomonas and Vibrio. Some of the microbiota that were identified are known to be pathogenic to fish: V.alginolyticus, V. anguillarum, and V. harveyi. Vibrio strains showed higher resistance to certain antibiotics com...

This study investigated the protective effect of polyb-hydroxybutyrate (PHB) on Chinese mitten crab, Eriocheir sinensis, zoea larvae challenged with pathogenic Vibrio anguillarum. PHB was delivered to the crab larvae through rotifer and Artemia bioencapsulation. Zoea 3 larvae were challenged with V. anguillarum at a final concentration of 10 5 CFU mL )1 . PHB-enriched rotifers and Artemia nauplii were added to the culture water 24 h prior to, upon and 24 h after challenge. The results confirmed that PHB could enhance the survival and growth of unexposed E. sinensis larvae. Moreover, PHB protected larvae from the pathogen as the larvae fed PHB-enriched live food showed the highest survival and development rate in all challenged groups (P < 0.05). Furthermore, larval performance was the best when PHB was delivered to the larvae 24 h before challenge (P < 0.05). In conclusion, our results indicate that PHB can be used as part of an effective strategy to protect E. sinensis larvae from V. anguillarum resulting in higher survival and better growth, especially when applied before the challenge.

ABSTRACTBackgroundVibrio cholerae,the causative agent of cholera, is a human pathogen that thrives in estuarine environments.V. choleraecompetes with neighboring microbes by the contact-dependent translocation of toxic effectors with the type VI secretion system (T6SS). Effector types are highly variable acrossV. choleraestrains, but all pandemic isolates encode the same set of distinct effectors. It is possible that acquisition of these effectors via horizontal gene transfer played a role in the development of pandemicV. cholerae.ResultsWe assessed the distribution ofV. choleraeT6SS loci across multipleVibriospecies. We showed that the fish-pathogenV. anguillarumencodes all threeV. choleraecore loci as well as two of the four additional auxiliary clusters. We further demonstrated thatV. anguillarumshares T6SS effectors withV. cholerae,including every pandemic-associatedV. choleraeeffector. We identified a novel T6SS cluster (Accessory Aux1) that is widespread inV. anguillarumand en...

Ferritin has been identified as the principal protein of iron storage and iron detoxification, playing a pivotal role for the cellular homeostasis in living organisms. However, recent studies in marine invertebrates have suggested its association with innate immune system. In the present study, one Ferritin subunit was identified from the gastropod Concholepas concholepas (CcFer), which was fully characterized by Rapid Amplification of cDNA Ends technique. Simultaneously, a challenge test was performed to evaluate the immune response against Vibrio anguillarum. The full length of cDNA Ccfer was 1030 bp, containing 513 bp of open reading frame that encodes to 170 amino acid peptide, which was similar to the Ferritin H subunit described in vertebrates. Untranslated Regions (UTRs) were identified with a 5 0 UTR of 244 bp that contains iron responsive element (IRE), and a 3 0 UTR of 273 bp. The predicted molecular mass of deduced amino acid of CcFer was 19.66 kDa and isoelectric point of 4.92. Gene transcription analysis revealed that CcFer increases against infections with V. anguillarum, showing a peak expression at 6 h post-infection. Moreover, a single nucleotide polymorphism was detected at À64 downstream 5 0 UTR sequence (SNP-64). Quantitative real time analysis showed that homozygous mutant allele (TT) was significantly associated with higher expression levels of the challenged group compared to wild (CC) and heterozygous (CT) variants. Our findings suggest that CcFer is associated to innate immune response in C. concholepas and that the presence of SNPs may involve differential transcriptional expression of CcFer.

Ocean acidification (OA) is one of the main consequences of increasing atmospheric carbon dioxide (CO2), impacting key biological processes of marine organisms such as development, growth and immune response. However, there are scarce studies on the influence of OA on marine invertebrates&#39; ability to cope with pathogens. This study evaluated the single and combined effects of OA and bacterial infection on the transcription expression of genes related to antioxidant system, antimicrobial peptides and pattern recognition receptors in the edible mussel Mytilus chilensis. Individuals of M. chilensis were exposed during 60 days at two concentrations of pCO2 (550 and 1200 μatm) representing respectively current and future scenario of OA and were then injected with the pathogenic bacterium Vibrio anguillarum. Results evidenced an immunomodulation following the OA exposure with an up-regulation of C-type Lectin and Mytilin B and a down-regulation of Myticin A and PGRP. This immunomodula...

A quaculture has continuously contained a potential risk of hypoxia. Because of this background, many studies on respiration and circulatory function of fish at hypoxic environment have been reported to date. Hypoxia influences the mortality of yellow tail infected with Enterococcus seriolicida. These findings suggest that exposing fish to hypoxia around the minimum requirement of environmental oxygen for long term may lead to a decrease in disease resistance in fish farming. Although studies on respiration and circulatory function at environmental oxygen below minimum requirement on fish have been reported, the immunological effect of environmental oxygen around the minimum requirement is unclear. Here, we examined the in vitro functions of rainbow trout (Oncorhynchus mykiss) leukocytes at environmental oxygen around the minimum requirement. Reactive oxygen generation and antibacterial activity of leukocyte cultured in low dissolved oxygen medium were decreased. The partial pressure of venous oxygen (PvO 2 ) of fish was decreased during hypoxia but it was quickly recovered by increasing of dissolved oxygen in environmental water. Furthermore, the mortality due to the experimental challenge with Vibrio anguillarum was reduced by expanding the ambient dissolved oxygen level. These results suggest that hypoxia around the minimum requirement of environmental oxygen adversely affects the functions of fish leukocytes. Our results also suggest that artificially increasing the PvO 2 can prevent fish from bacterial infectious diseases in aquaculture.