Resistance of Argopecten purpuratus scallop larvae to vibriosis is associated with the front-loading of immune genes and enhanced antimicrobial response.

Mass mortality events caused by vibriosis have emerged in hatchery-reared scallop larvae from Chile, threatening scallop aquaculture. In an attempt to mitigate this emerging infectious disease and provide candidates for marker-assisted selective breeding, we tested here the existence of a genetic component of Argopecten purpuratus scallop resistance to the pathogen Vibrio bivalvicida. Through a dual RNA-seq approach we analyzed the basal transcriptome and the transcriptional response to infection in two resistant and two susceptible families as well as the pathogen transcriptomic response to host colonization. The results highlighted a genetic basis in the resistance of scallop larvae to the pathogen. The Vibrio response was characterized by a general metabolic adaptation to the host environment, along with several predicted virulence factors overexpressed in infected scallop larvae with no difference between resistant and susceptible host phenotypes. On the host side, several biological processes were enriched in uninfected resistant larvae. Within these enriched categories, immune-related processes were overexpressed, while morphogenesis, biomineral tissue development, and angiogenesis were under expressed. Particularly, genes involved in immune recognition and antimicrobial response, such as lipopolysaccharide-binding proteins (LBPs), lysozyme, and bactericidal permeability-increasing protein (BPI) were overexpressed in uninfected resistant larvae. As expected, immune-related biological processes were enriched in Vibrio-infected larvae, but they were more numerous in resistant larvae. Overexpressed immune genes in response to infection included several Toll-like receptors, TNF and NF-κB immune signaling genes, and the antimicrobial peptide Big defensin ApBD1. Results strongly suggest that both a front-loading of immune genes and an enhanced antimicrobial response to infection contribute to the resistance, while pathogen infective strategy does not discriminate between host phenotypes. Overall, early expression of host immune genes appears as a strong determinant of the disease outcome that could be used in marker-assisted selective breeding.

Probiotic and Immunomodulatory Activity of Marine Yeast Yarrowia lipolytica Strains and Response Against Vibrio parahaemolyticus in Fish.

Yarrowia lipolytica has been widely used in food industry but scarcely explored as probiotics. Thus, the aims of this study were to characterize in vitro the probiotic potential, antioxidant capacity, and antimicrobial activity of the marine yeast Y. lipolytica D-1 and N-6 strains. Dietary administration effect was evaluated in vivo on immunological parameters in serum, skin-mucus, intestine, and fish leukocytes upon challenge with Vibrio parahaemolyticus. The results showed that Y. lipolytica D-1 and N-6 strains grew with NaCl or bile salts but were sensitive to low pH. Each of the Y. lipolytica strains had a distinctive antioxidant capacity and fatty acid profile, but their antimicrobial activity was similar against fish bacterial pathogens. Fish (Lutjanus peru) supplemented with Y. lipolytica strains showed normal intestinal morphology, high IgM levels, and antioxidant enzyme activities. Immune-related genes were modulated in fish fed Y. lipolytica in a strain-dependent fashion. In addition, leucocytes from fish fed Y. lipolytica challenged with V. parahaemolyticus increased innate immune and antioxidant parameters compared with the control groups. In conclusion, the marine yeast Y. lipolytica D-1 and N-6 strains may be potential probiotics for fish by exerting free-radical scavenging, antimicrobial activity, and improved immune-protective responses against V. parahaemolyticus infection.

Isolation of Harveyi clade Vibrio spp. collected in aquaculture farms: How can the identification issue be addressed?

Aquaculture is a fast growing industry with its development hampered by bacterial diseases. Vibriosis caused by Harveyi clade strains is known for causing heavy loss especially in shrimp aquaculture farms. For farm treatment and pathogen spread management, veterinarians and researchers need reliable bacterial identification tools. A range of identification methods have been presented for Vibrio spp. in recent literature but little feedback on their performance have been made available to this day. This study aims at comparing Vibrio spp. identification methods and providing guidance on their use. Fifty farms were sampled and bacterial colonies were isolated using specific culture media before microscopic analysis and genomic profiling using ERIC-PCR. A preliminary identification step was carried out using MALDI-ToF mass spectrometry. Four methods were compared for strain identification on 14 newly isolated Harveyi clade Vibrio spp. strains: whole genome sequencing (digital DNA DNA Hybridization (dDDH)), 5 MLSA schemes, ferric uptake regulation (fur) and lecithin-dependent haemolysin (ldh) single gene based identification methods. Apart from dDDH which is a reference method, no technique could identify all the isolates to the species level. The other tested techniques allowed a faster, cheaper but sub genus clade identification which can be interesting when absolute precision is not required. In this regard, MALDI-ToF and fur based identification seemed especially promising.

Identification and characterization of outer membrane vesicles from the fish pathogen Vibrio ordalii.

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.

First screening report of immune and protective effect of non-toxic Jatropha vernicosa stem bark against Vibrio parahaemolyticus in Longfin yellowtail Seriola rivoliana leukocytes.

In México, the infusion of Jatropha vernicosa stem bark has been used in folk medicine for many clinical situations, but no reports were available about this particular species of Jatropha in fish of mammals. In this first screening report, the phytochemical, antioxidant profile and antimicrobial properties of aqueous J. vernicosa stem bark extract were explored against Vibrio parahaemolyticus, an opportunist fish pathogen. To evaluate the cytotoxicity and immunological effect for the possible application of aqueous J. vernicosa stem bark in aquaculture, this study assessed it by using Longfin yellowtail Seriola rivoliana leukocytes. The results showed that phytochemical composition of the J. vernicosa extract was rich in phenol, flavonoid, saponin, and coumarin compounds. The antioxidant capacity of hydroxyl radical and superoxide anion scavenging activities, iron-chelation activity and ß-carotene bleaching coupled to linoleic acid showed that J. vernicosa extracts had a moderate antioxidant effect compared with synthetic antioxidants (BHT, BHA and EDTA). No adverse effects were observed on spleen leukocytes (viability > 98%). Interestingly, J. vernicosa stem bark extract has immunostimulant and antioxidant effects, increasing phagocytosis, respiratory burns activity, and nitric oxide production, as well as superoxide dismutase and catalase activities. Additionally, J. vernicosa extract increased pro-inflammatory cytokine IL-1ß and suppressed anti-inflammatory IL-10 gene expression upon stimuli and V. parahaemolyticus challenge. Finally, the data confirms that J. vernicosa stem bark extract is non-cytotoxic, rich in bioactive compounds with antioxidant effects, capable of enhancing the immune system in leukocytes and with great potential to fight against opportunistic diseases, such as vibriosis in fish.

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii.

Vibrio ordalii is an extracellular, Gram-negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo-LM-18 and ATCC 33509T in the fish-cell lines SHK-1 and CHSE-214. Confocal microscopy revealed Vo-LM-18 and ATCC 33509T inside cytoplasm in both fish-cell lines at 4 hr post-inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo-LM-18 and ATCC 33509T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.

Susceptibility of Pacific white snook Centropomus viridis to Vibrio species.

To examine the pathogenicity of Vibrio strains, several doses of Vibrio harveyi (CAIM 1622 and CAIM 1508), Vibrio ponticus (CAIM 1751) and Vibrio anguillarum (CAIM 8) were used to challenge Pacific white snook Centropomus viridis Lockington, 1877 juveniles, and survival, gross signs and histological lesions were observed. Susceptibility of pathogenic vibrios CAIM 1508 and CAIM 1751 to antibiotics used in aquaculture was also evaluated. The growth ability of the tested strains was not related to their pathogenicity. One of the V. harveyi strains (CAIM 1508) was the most virulent, causing per-acute septicaemia in C. viridis even at a low dose (1.4 × 104 CFU g-1). Although the V. ponticus strain (CAIM 1751) was less virulent, this is the first report of it as a pathogen of white snook. Fish challenged with V. ponticus displayed external, generalized haemorrhaging. Necrosis of the digestive tract and intravascular haemosiderosis were the most remarkable histological lesions in fish challenged with both strains. Multifocal necrosis of the internal organs and bacterial masses was also observed. The lowest minimum inhibitory concentration of the pathogenic strains (CAIM 1508 and CAIM 1751) was calculated for enrofloxacin (20 and 10 µg ml-1, respectively), and both bacteria were resistant to amoxicillin, ampicillin and trimethoprim-sulfamethoxazole.

Variations in the immune and metabolic response of proactive and reactive Sparus aurata under stimulation with Vibrio anguillarum vaccine.

Environmental insults, such as exposure to pathogens, modulate the behavioural coping style of animals to stressors, and repeated exposure to stressful environments may lead to species-specific infection phenotypes. To analyse the influence of stress behavioural phenotypes on immune and metabolic performance, gilthead sea bream (Sparus aurata L.) were first screened for proactive and reactive coping styles. Once characterized, both behavioural phenotypes fish groups were bath vaccinated with bacterin from Vibrio anguillarum, an opportunistic widespread pathogen of fish. Gills and liver were sampled at 0 (control group), 1, 3 and 7 days post-vaccination. Immune-, oxidative stress- and metabolic-related transcripts (il1ß, tnfα, igm, gpx1, sod, cat, lpl, ghr1 and ghr2), metabolic endpoints (glucose, cholesterol and triglycerides), hepatic health indicators (aspartate aminotransferase, alanine transaminase and alkaline phosphatase), oxidative stress status (esterase activity, total antioxidant capacity and total oxidative status) and stress biomarkers (cortisol) were determined. Present results indicate that screening for coping styles in the gilthead sea bream segregated the two distinct phenotypes as expected: proactive and reactive. Results also indicate that under bath vaccination proactive fish show high immune response and lower metabolism, whereas reactive fish show low immune and higher metabolic responses.

Caspase -1, -3, -8 and antioxidant enzyme genes are key molecular effectors following Vibrio parahaemolyticus and Aeromonas veronii infection in fish leukocytes.

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In order to get insights into the caspase gene family and antioxidant enzymes in Totoaba macdonaldi during bacterial infection, an in vitro assay was performed involving three different types of caspases (Casp-1, Casp-3 and Casp-8) and antioxidant enzymes (catalase, gluthathione peroxidase 1 and 4) after Vibrio parahaemolyticus and Aeromonas veronii infection, using head-kidney and spleen leukocytes from the teleost fish totoaba at 12 and 24 h post-exposure. Characterization of caspases by bioinformatics analyses showed that TmCas-1, TmCas-3 and TmCas-8 shared overall sequence identities of 82-61%, 85-97% and 77-63%, respectively, with other teleost fish. Caspase-1, -3 and -8 proteins revealed a conserved penta-peptide sequence at the catalytic site and three amino acid residues involved in the catalysis (H, G and C), as well as two conserved domains. The expression levels of the three caspases were detected in a wide range of fish tissues; however, they varied among tissues and caspases, which were highly up-regulated in immune organs, such as head-kidney, liver and/or spleen. The pathogen-induced gene expression pattern revealed two interesting facts; first, that the expression of all the caspase genes and antioxidant enzyme genes evaluated in this study were strongly induced following V. parahaemolyticus infection; second, these up-regulations reached a maximum level at 24 h post-infection in head-kidney whereas in spleen leukocytes, it was observed at 6-h post-infection. In conclusion, based on these observations, the acute toxic effects of V. parahaemolyticus are associated to cell death and release of free radicals. This information provides a better understanding of the effects and nature of early immune response against common bacterial infections in totoaba leukocytes.

Proteomic profiling of integral membrane proteins associated to pathogenicity in Vibrio parahaemolyticus strains.

Vibrio parahaemolyticus has been recognized as the causal agent of early mortality syndrome and is currently considered an emerging shrimp disease causing losses of millions in the aquaculture industry. Integral membrane proteins are widely recognized as pathogenicity factors involved in essential mechanisms for V. parahaemolyticus infection, which makes them attractive as therapeutic targets. However, their physico-chemical properties and weak expression has resulted in under-representation of these proteins in conventional bottom-up proteomics, making integral membrane proteomics a challenging task. Integral membrane proteins from a bacterial strain isolated from the hepatopancreases of white shrimp with early mortality syndrome and identified by 16S rRNA sequencing as V. parahaemolyticus and an ATCC strain that is pathogenic for humans were obtained by a sequential extraction method and subjected to relative quantification and identification by isobaric Tags for Relative and Absolute Quantitation. A homology database search resulted in identification of more than two hundred proteins, 35 of which are recognized as pathogenic factors showed statistically significant differential accumulation between the strains. These proteins are mainly associated with adherence, secretion systems, cell division, transport, lysogenization, movement and virulence. Identification of pathogenicity-related proteins in V. parahaemolyticus provides valuable information for developing strategies based on molecular mechanisms that inhibit these proteins, which may be useful therapeutic targets for assisting the shrimp and aquaculture industry.

Functional diets modulate the acute phase protein response in Oncorhynchus mykiss subjected to chronic stress and challenged with Vibrio anguillarum.

The acute phase response to pathogens alters the production of proinflammatory cytokines that, in turn, activate the synthesis of acute phase proteins. These proteins neutralize, prevent, and indicate tissue damage, thereby influencing the specific immune response and allowing the organism to regain homeostasis. Functional diets based in pre- and probiotics are used in aquaculture to improve fish health and resistance to diseases, but there is an information gap on the mechanisms involved in these effects and if these diets are efficient when fish are raised under high stocking densities. This study aimed an evaluation of the acute phase response in Oncorhynchus mykiss fed functional diets supplemented with pre- and probiotics (i.e. mannan-oligosaccharides and Saccharomyces cerevisiae, respectively) and challenged by either Vibrio anguillarum or chronic stress via maintenance under high stocking densities. For this, the relative expression of acute phase response related genes in liver, and of inflammatory response related genes in head kidney was evaluated by RT-qPCR. The supplemented diets differentially modulated the acute phase protein response to the assessed challenge conditions, specifically evidencing an overexpression of the genes HAPT, SAA, LECT2, and IL-1ß under chronic stress and of HAPT, IL-1ß, IL8, and LECT2 at 24 h post-challenge with V. anguillarum. The observed early-stage regulation of acute phase proteins and of the immune response by the probiotic S. cerevisiae and by prebiotic mannan-oligosaccharides suggests that both supplements have high immunostimulatory potentials for fish farmed under high stocking densities.

Dietary yeast Sterigmatomyces halophilus enhances mucosal immunity of gilthead seabream (Sparus aurata L.).

A yeast was isolated from hypersaline sediments, grown and phylogenetically characterized as Sterigmatomyces halophilus strainN16. The dietary administration of this yeast was studied for its effect on skin mucosal immune and antioxidant status of gilthead seabream (Sparus aurata L.). Fish were fed a commercial diet (control, non-supplemented diet), or the same commercial diet supplemented with 0.55% or 1.1% of yeast for 15 and 30 days. One month after the end of the trial, fish from all treatments were intraperitoneally injected with pathogenic Vibrio parahaemolyticus and further fed with the same diets for one week, after which fish were also sampled. Significant increases were observed in the immune activities determined in the fish fed the yeast supplemented diets compared with the values recorded in mucus of fish from the control group. The expression levels of trypsin (one of the main digestive enzymes) and several immune-related genes (IL-1ß, TNF-α, IgM, C3 and lysozyme) were also evaluated by real-time PCR in intestine and skin. Interestingly, trypsin gene expression in intestine was up regulated in both experimental diets compared with the control group, particularly in fish fed with 0.55% of S. halophilus at any time of the experimental trial. Immune-related genes in intestine and skin were strongly expressed principally in fish fed with 0.55% of S. halophilus for 15 days and 1.1% for 30 days and after infection, respectively. The present results suggest that the yeast S. halophilus can be considered as a novel fish immunostimulant. The excellent potential of marine microorganisms isolated from extreme environments with beneficial properties for fish is discussed.

Enhancing gilthead seabream immune status and protection against bacterial challenge by means of antigens derived from Vibrio parahaemolyticus.

In an attempt to control the proliferation of the pathogenic bacterium Vibrio parahaemolyticus in gilthead seabream (Sparus aurata), the immunostimulant effect of lysate and ToxA from this bacterium was evaluated. Fish were intraperitoneally injected twice (first injection, day 1 of the experiment; second injection, day 7) and sampled after one week (on days 8 and 15). Afterwards, all fish specimens were experimentally infected with V. parahaemolyticus and mortality was recovered for 1 week. Fish injected with lysate, ToxA and phosphate buffer saline (control) showed 100%, 50% and 0% survival, respectively, when challenged with the pathogen. Skin mucus immune parameters and immune-related gene expression in skin and spleen were also evaluated. The results showed that mucus immune parameters were enhanced in the lysate and ToxA groups compared with the values obtained for fish from the control group. Expression of IL-1ß, TNF-α, C3 and IgM genes was significantly up-regulated in the lysate and ToxA groups, principally after infection with the bacterium. Interestingly, TLR5 gene expression increased in fish immunized with lysate. The most prominent histological characteristic in gut from infected fish was the presence of a great number of intraepithelial leucocytes as well as inflammation of the submucosa, while severe hydropic degeneration and hemosiderosis were detected in liver from infected fish. Injection of lysate or ToxA had a protective effect against the deleterious consequences of subsequent infection with V. parahaemolyticus in gut and liver. The findings underline the potential of lysate and ToxA as potent preventive antigens against this kind of vibriosis.

Evaluation of ToxA and Vibrio parahaemolyticus lysate on humoral immune response and immune-related genes in Pacific red snapper.

Immunogenicity of ToxA and Vibrio parahaemolyticus lysate was evaluated in a double immunostimulation scheme in Pacific red snapper after V. parahaemolyticus infection. Three groups of Pacific red snapper were intraperitonealy (i.p.) injected with phosphate-buffered saline (PBS group), ToxA of V. parahaemolyticus (ToxA-Vp group) or V. parahaemolyticus lysate (lysate-Vp group) (first injection, day 1; second injection, day 7). Fish were subsequently infected with live V. parahaemolyticus. Humoral immune parameters in skin mucus and serum were evaluated on days 1, 7, 8 and 14 days post-immunostimulation and 7 days post-infection. Moreover expression of immune-related genes was quantified by real time PCR in head-kidney leukocytes, spleen, liver, and intestine. The ToxA-Vp-treated group showed a higher anti-protease and catalase activity in skin mucus when compared with the PBS group. Measurements of SOD and CAT activities showed an increment in both activities a day after the second boost with ToxA-Vp or lysate-Vp. Interestingly, IgM levels in mucus and transcripts were enhanced followed the ToxA-Vp treatment even after challenge. Furthermore, IL-1ß was strongly expressed in all analyzed cell or tissues followed ToxA-Vp or Vp-lysate treatments. Finally, SOD and CAT gene expression was up-regulated in fish immunostimulated with either treatment ToxA-Vp or lysate-Vp, mainly after infection in head-kidney leukocytes and intestine. This is the first study where the effects of ToxA from V. parahaemolyticus in the immune system of Pacific red snapper was evaluated. These results suggest that ToxA-Vp would positively affect humoral immune response and up-regulate expression of genes involved in the immune system function; and could help in the control of V. parahaemolyticus infection in Pacific red snapper Lutjanus peru, an economic important fish in Mexico.

Survival behaviour and virulence of the fish pathogen Vibrio ordalii in seawater microcosms.

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.

Prevalence and Distribution of Vibrio spp. in Wild Aquatic Birds of the Southern Caribbean Sea, Venezuela, 2011-12.

Vibrio spp. are associated with waterbirds mainly in temperate latitudes. We evaluated the prevalence and distribution of Vibrio spp. from fecal samples of resident and migratory aquatic birds collected during October 2011 and March 2012 at two coastal sites in the tropical southern Caribbean Sea. We amplified DNA by PCR in 40% of samples, resulting in 47% and 36% estimated prevalence for resident and migratory birds in Cuare Wildlife Refuge, and 33% and 44% in Margarita Island, respectively. We found nontoxigenic Vibrio cholerae in Cuare Wildlife Refuge with a higher prevalence in resident birds (18%). Our PCR results for Vibrio and V. cholerae were not significantly different between sites or bird migratory status. The 16S rRNA phylogenetic analysis sequences from fecal samples from Cuare Wildlife Refuge were highly similar to V. cholerae and Vibrio vulnificus , whereas sequences from Margarita Island samples formed clusters with species related to the Harveyi clade. Our findings indicate that several species of Vibrio are common in aquatic birds along the southern Caribbean Sea and contribute to our understanding of the role of birds as possible reservoirs of potentially pathogenic bacteria.

Iron assimilation and siderophore production by Vibrio ordalii strains isolated from diseased Atlantic salmon Salmo salar in Chile.

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.

Effects of methanolic macroalgae extracts from Caulerpa sertularioides and Ulva lactuca on Litopenaeus vannamei survival in the presence of Vibrio bacteria.

Macroalgae are potentially excellent sources of highly bioactive secondary metabolites that are useful for the development of new functional ingredients. This study was conducted to determine whether methanolic extracts from Caulerpa sertularioides and Ulva lactuca macroalgae might be possible alternatives for the prevention of shrimp vibriosis, which is caused by Vibrio parahaemolyticus and Vibrio alginolyticus. Macroalgae extracts prepared with methanol as the solvent were evaluated for antibacterial activity with the microplate method. The extracts' effects on the mortality of juvenile Litopenaeus vannamei were evaluated at doses of 150 and 300 mg L(-1). Two independent assays for V. parahaemolyticus and V. alginolyticus were performed. The methanolic extract of C. sertularioides exhibited activity against V. parahaemolyticus and V. alginolyticus, and it had minimal inhibitory concentrations of <1000 and < 1500 µg mL(-1), respectively. L. vannamei mortality in the presence of both The methanolic extract of C. sertularioides exhibited activity against V. parahaemolyticus and V. alginolyticus, and it had minimal inhibitory concentrations of <1000 and <1500 µg mL(-1), respectively. and V. alginolyticus bacteria significantly decreased after treatment with 300 mg L(-1) C. sertularioides methanolic extract.

Isolation and identification of Vibrio toranzoniae associated with diseased red conger eel (Genypterus chilensis) farmed in Chile.

The present study deals with the first isolation of Vibrio toranzoniae from cultured red conger eel (Genypterus chilensis). During the summer season of 2011, mortalities were observed in young red conger eel at one aquaculture experimental rearing system in Quintay, Valparaiso, Chile. The microbiological analysis of the diseased fish resulted in the isolation of three dominant and representative isolates, designated as R.17, R.18 and R.19, which were obtained from gill, fin and external lesions from three different fish, respectively. All isolates were identified as V. toranzoniae by means of a polyphasic taxonomic approach, including phenotypic characterization, sequencing of 16S rRNA and housekeeping genes, and DNA-DNA hybridization. Inoculation of a representative strain (R18) in turbot as model fish species demonstrated the pathogenic potential for fish of the Chilean isolates. Results obtained indicate that the geographical and host distribution of V. toranzoniae is wider than expected, and that this species may have negative incidence in the culture of marine organisms.