FrpA is the outer membrane piscibactin transporter in Vibrio anguillarum: structural elements in synthetic piscibactin analogues required for transport.

Piscibactin (Pcb) is a labile siderophore widespread among Vibrionaceae. Its production is a major virulence factor of some fish pathogens such as Photobacterium damselae subsp. piscicida and Vibrio anguillarum. Although FrpA was previously suggested as the putative outer membrane transporter (OMT) for ferri-piscibactin, its role in piscibactin uptake was never demonstrated. In this work, we generated mutants of V. anguillarum defective in FrpA and analyzed their ability to use piscibactin as iron source. The results showed that inactivation of frpA completely disables piscibactin utilization, and the original phenotype could be restored by gene complementation, confirming that FrpA is the OMT that mediates ferri-Pcb uptake. Additionally, the ability of several Pcb thiazole analogues, with different configurations at positions 9, 10, and 13, to be internalized through FrpA, was evaluated measuring their ability to promote growth under iron deficiency of several indicator strains. The results showed that while those analogues with a thiazole ring maintain almost the same activity as Pcb, the maintenance of the hydroxyl group present in natural piscibactin configuration at position C-13 is crucial for Fe3+ chelation and, in consequence, for the recognition of the ferri-siderophore by the cognate OMT. All these findings allowed us to propose a Pcb analogue as a good candidate to vectorize antimicrobial compounds, through the Trojan horse strategy, to develop novel compounds against bacterial fish diseases.

Inhibition of lysozyme lytic activity by Ivy derived from Photobacterium damselae subsp. piscicida.

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

In vitro hemocyte phagocytosis activation after experimental infection of common octopus, Octopus vulgaris (Cuvier, 1797) with Photobacterium damselae subsp. piscicida or Vibrio alginolyticus at different temperatures and infection routes.

Due to the fast growth rate, the short life cycle, the high market price and the high food conversion efficiency, O. vulgaris is considered as a good candidate for aquaculture. One of the prerequisites for the successful integration of new species, such as octopi, into industrial-scale production, is the knowledge of the pathological conditions that may arise, with emphasis on infectious diseases caused by microorganisms and para-sites transmitted through wild populations, especially for the farmed organisms cul-tured in cages in proximity to teleost fish. The main objectives of this study were to investigate the sensitivity of common octopus to experimental infection with pathogenic bacteria, to assess the activation of hemocytes and more specifically their phagocytic activity after infection and to associate sensitivity of the species and phagocytic activity of hemocytes to temperature changes, route of infection and pathogen. Common octopus individuals were intramuscularly and intravenously infected with either Photobacterium damselae subsp. piscicida or Vibrio alginolyticus. The hemocyte phagocytosis activation in vitro at two temperatures (21 ± 0.5 °C and 24 ± 0.5 °C) was studied, in an effort to relate these aspects to climate change. Hemolymph was withdrawn on days 0, 3 and 7 post infections/injections. Number of circulating hemocytes/ml hemolymph, phagocytosis ability and Phagocytosis Particle Binding Intensity index were determined. Correlations between hemocytes and bodyweight and between hemocytes and phagocytosis ability were also determined. No mortalities were recorded irrespective of pathogen, route of infection and temperature employed. Circulating hemocytes in control specimens ranged between 1.60x105 hemocytes ml-1 hemolymph to 20.02x105 hemocytes/ml hemolymph at both experiments and temperatures. The interrelation between octopi weight and circulating hemocytes showed that natural fluctuations, age, maturity stage and temperature may affect this relationship. Rise of temperature influenced phagocytosis which seemed to be route of infection, time-point and pathogen related. Specimens infected with Photobacterium damselae subsp. piscicida showed decreased phagocytosis with rise of temperature while when Vibrio alginolyticus was used, phagocytosis activity increased, in most cases. Temperature also played an important role in the correlation between the circulating hemocytes and phagocytosis activity, as at lower temperatures a negative strong correlation was observed. The results prompted us to calculate the activation index. This index showed that temperature is an important factor in hemocyte activation since for Photobacterium damselae subsp. piscicida infected specimens, hemocytes were more activated at 21 ± 0.5℃ instead of 24 ± 0.5℃, and the opposite observed for Vibrio alginolyticus samples and only later post-infection. Comparing the phagocytosis ability results with those obtained from Particle Binding Intensity index important differences concerned mainly confidence levels. The use of phagocytosis ability instead of PBI index provides more accurate results.

A Highly Unstable and Elusive Plasmid That Encodes the Type III Secretion System Is Necessary for Full Virulence in the Marine Fish Pathogen Photobacterium damselae subsp. piscicida.

The marine bacterium Photobacterium damselae subsp. piscicida (Pdp) causes photobacteriosis in fish and important financial losses in aquaculture, but knowledge of its virulence factors is still scarce. We here demonstrate that an unstable plasmid (pPHDPT3) that encodes a type III secretion system (T3SS) is highly prevalent in Pdp strains from different geographical origins and fish host species. We found that pPHDPT3 undergoes curing upon in vitro cultivation, and this instability constitutes a generalized feature of pPHDPT3-like plasmids in Pdp strains. pPHDPT3 markers were detected in tissues of naturally-infected moribund fish and in the Pdp colonies grown directly from the fish tissues but were undetectable in a fraction of the colonies produced upon the first passage of the primeval colonies on agar plates. Notably, cured strains exhibited a marked reduction in virulence for fish, demonstrating that pPHDPT3 is a major virulence factor of Pdp. The attempts to stabilize pPHDPT3 by insertion of antibiotic resistance markers by allelic exchange caused an even greater reduction in virulence. We hypothesize that the existence of a high pressure to shed pPHDPT3 plasmid in vitro caused the selection of clones with off-target mutations and gene rearrangements during the process of genetic modification. Collectively, these results show that pPHDPT3 constitutes a novel, hitherto unreported virulence factor of Pdp that shows a high instability in vitro and warn that the picture of Pdp virulence genes has been historically underestimated, since the loss of the T3SS and other plasmid-borne genes may have occurred systematically in laboratories for decades.

Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication.

Apoptosis-inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram-negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single-chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc-metalloprotease moiety that cleaves the NF-kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase-thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins.

Protective efficacy of four heat-shock proteins as recombinant vaccines against photobacteriosis in Asian seabass (Lates calcarifer).

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.

Inclusion of dietary Ulva ohnoi 5% modulates Solea senegalensis immune response during Photobacterium damselae subsp. piscicida infection.

Macroalgae represent valuable sources of functional ingredients for fish diets, and the influence of supplemented aquafeeds on growth performance has been studied for some fish and seaweed species. In the present work, the potential immunomodulation exerted by U. ohnoi (5%) as dietary ingredient was investigated in Senegalese sole. After feeding with the experimental diets for 90 d, fish immune response before and after challenge with Photobacterium damselae subsp. piscicida (Phdp) was assessed. In absence of infection, systemic immune response was not modified by 5% U. ohnoi dietary inclusion for 90 d. Thus, no differences in liver and head kidney immune gene transcription or serum lysozyme, peroxidase, antiprotease and complement activities were observed based on the diet received by Senegalese sole specimens. Regarding mucosal immune parameters, no changes in gene transcription were detected in the skin and gills, whilst only tnf, cd4 and cd8 were significantly up-regulated in the intestine of fish fed with U. ohnoi, compared to the values obtained with control diet. On the contrary, when S. senegalensis specimens were challenged with Phdp, modulation of the immune response consisting in increased transcription of genes encoding complement (c1q4, c3, c9), lysozyme g (lysg), tumor necrosis factor alpha (tnfα) as well as those involved in the antioxidant response (gpx, sodmn) and iron metabolism (ferrm, hamp-1) was observed in the liver of fish fed with U. ohnoi. In parallel, decreased inflammatory cytokine and complement encoding gene transcription was displayed by the spleen of fish receiving the algal diet. Though mortality rates due to Phdp challenge were not affected by the diet received, lower pathogen loads were detected in the liver of soles receiving U. ohnoi diet. Further research to investigate the effects of higher inclusion levels of this seaweed in fish diets, feeding during short periods as wells as to assess the response against other pathogens needs to be carried out.

Genotypic diversity, and molecular and pathogenic characterization of Photobacterium damselae subsp. piscicida isolated from different fish species in Taiwan.

Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%-100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.

Outer membrane protein FrpA, the siderophore piscibactin receptor of Photobacterium damselae subsp. piscicida, as a subunit vaccine against photobacteriosis in sole (Solea senegalensis).

Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main infectious diseases affecting cultured fish in Mediterranean countries. Diverse vaccine formulations based in the use of inactivated bacterial cells have been used with unsatisfactory results, especially in newly cultured species like sole (Solea senegalensis). In this work, we describe the use of the outer membrane receptor (FrpA) of the siderophore piscibactin produced by Pdp as a novel subunit vaccine against photobacteriosis. FrpA has been cloned and expressed in Escherichia coli under an arabinose-inducible promoter. A recombinant protein (rFrpA) containing the pelB localization signal and a His tag was constructed to obtain a pure native form of the protein from E. coli outer membranes. The immunogenicity of rFrpA, and its protective effect against photobacteriosis, was tested by i.p. injection of 30  µg of the protein, mixed with Freund's adjuvant, in sole fingerlings with two immunizations separated by 30 days. Results showed that using either pure rFrpA or whole cells as immobilized antigens in ELISA assays, rFrpA induces the production of specific antibodies in sole. An experimental infection using fish vaccinated with rFrpA or formalin-killed whole cells of Pdp showed that both groups were protected against Pdp infection at similar levels, with no significant differences, reaching RPS values of 73% and 79%, respectively. Thus, FrpA constitutes a promising antigen candidate for the development of novel more effective vaccines against fish photobacteriosis.

Detection of specific immune response in sole (Solea senegalensis) against Photobacterium damselae subsp. piscicida antigens.

The pathogenic bacteria Photobacterium damselae subsp. piscicida affects the development of Solea senegalensis culture. Vaccines made with inactivated cells have produced a relative protection against the sickness, however the administration of subcellular and purified antigens as vaccine could increase the effectiveness of the immune response. Thus, the aim of this work was the determination of antigens of P. damselae subsp. piscicida involved in the specific immune response of S. senegalensis. Fish were immunized by intraperitoneal injection (i.p.) with inactivated extracellular polymeric substances (ECP) and whole cells of P. damselae subsp. piscicida, and Freund's incomplete adjuvant. Two months later fish were boosted with the same antigens. Serum from fish was collected to determine by ELISA the title of antibodies against subcellular fractions of bacteria (ECP, capsule, outer membrane proteins, O antigen and formalized whole cells). Significant differences were found between control and immunized fish, but differences between first immunization and booster were only found for O antigen and capsule. Western blots derived from 2D-PAGE of ECP and Outer Membrane Proteins (OMP), using sole immunized serum, detected two high reactive antigens from ECP. Proteins were identified, by mass spectrometry, as ATP-dependent metalloprotease and Telurite resistance proteins. In the case of OMP, three antigenic proteins were detected and identified as Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related.

Fish photobacteriosis-The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida.

MALDI-TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on-target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24-hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48-hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI-TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate-dependent.

Physiopathological responses of sole (Solea senegalensis) subjected to bacterial infection and handling stress after probiotic treatment with autochthonous bacteria.

This study aimed to evaluate the protective effects of four autochthonous bacteria isolated from juvenile sole (Solea senegalensis) intestine as dietary probiotic supplement against bacterial pathogen infection and handling/transport stressors. Growth performance and immune responses were evaluated after 85 days of feeding trial. Sole (IBW = 16.07 ±â€¯0.11 g) were fed six experimental diets, a control diet (CTRL, without the dietary probiotic supplementation), and five diets supplemented with probiotic bacteria: PB1 (Shewanella hafniensis), PB2 (Enterococcus raffinosus), PB3 (Shewanella hafniensis + Arthrobacter soli), PB4 (Pseudomonas protegens + Arthrobacter soli) and PB5 (Shewanella hafniensis + Arthrobacter soli + Enterococcus raffinosus). All bacteria were selected based on their in vitro antimicrobial activity. After the growth trial, fish were submitted to a stress factor (transport) and then each dietary group was divided in two additional groups: non-infected (placebo) and infected with Photobacterium damselae subsp. piscicida. Immune and antioxidant responses were evaluated at day 10 post-infection. In infection trial A, fish were infected on the same day of transport, whereas in trial B fish were infected after a 7-day recovery from the transport stress. At the end of the feeding trial, fish fed with PB2 and PB4 showed lower final body weight when compared with the other dietary groups. Respiratory burst activity and nitric oxide production were not affected by probiotic supplementation. Fish fed with PB5 presented lower peroxidase activity compared to CTRL. Lysozyme and alternative complement pathway activity (ACH50) showed no significant differences between treatments. The innate immune responses were significantly affected after handling stress and bacterial infection. In trial A, the ACH50 levels of infected fish were significantly lower than the placebo groups. On the other hand, in trial B fish infected with Pdp demonstrated higher ACH50 levels when compared to placebos. Peroxidase levels were strongly modulated by bacterial infection and handling stress. In trials A and B, infection had a clear downgrade effect in peroxidase levels. Lipid peroxidation, catalase, glutathione S-transferase and glutathione reductase were altered by both bacterial infection and transport. Overall, dietary probiotic supplementation did not influence growth performance of sole. The immune and oxidative defenses of sole responded differently to infection depending on the probiotic and the synergy between pathogen infection and transport.

De novo transcriptome analysis of immune response on cobia (Rachycentron canadum) infected with Photobacterium damselae subsp. piscicida revealed inhibition of complement components and involvement of MyD88-independent pathway.

Cobia, Rachycentron canadum, one of the most important aquatic species in Taiwan, has suffered heavy losses from Photobacterium damselae subsp. piscicida, which is the causal agent of photobacteriosis. In this study, the transcriptomic profiles of livers and spleens from Pdp-infected and non-infected cobia were obtained for the first time by Illumina-based paired-end sequencing method with a focus on immune-related genes. In total, 164,882 high quality unigenes were obtained in four libraries. Following Pdp infection, 7302 differentially expressed unigenes from liver and 8600 differentially expressed unigenes from spleen were identified. Twenty-seven of the differently expressed genes were further validated by RT-qPCR (average correlation coefficient 0.839, p-value <0.01). Results indicated a negative regulation of complement components and increased expression of genes involved in MyD88-independent pathway. Moreover, a remarkable finding was the increased expression of IL-10, implying an inadequacy of immune responses. This study not only characterized several putative immune pathways, but also provided a better understanding of the molecular responses to photobacteriosis in cobia.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Transcription of immune related genes in Solea senegalensis vaccinated against Photobacterium damselae subsp. piscicida. Identification of surrogates of protection.

Solea senegalensis is a flatfish with a great potential for aquaculture, but infectious diseases restrict its production, being this fish species highly susceptible to Photobacterium damselae subsp. piscicida (Phdp) infections. A better understanding of the mechanisms related to fish immune response is crucial for the development of effective approaches in disease management. In the present work, transcriptional changes of immune related genes have been evaluated in farmed S. senegalensis specimens vaccinated against Phdp by intraperitoneal injection (IP) and immersion (IM). IP fish showed higher antibody levels and increased transcription of genes encoding lysozyme C1, complement factors involved in the classical pathway and components involved in the opsonization and the limitation of free iron availability, all of them facilitating the faster elimination of the pathogen and promoting higher RPS after the infection with Phdp. The results of this study seem to support a different intensity of the specimens immune response in the head kidney. Analysis of the immune response in 15 day post-challenged fish showed up-regulation of genes involved in all stages of S. senegalensis immune response, but especially those genes encoding proteins related to the innate response such as complement, lysozyme and iron homeostasis in the head kidney. On the other hand, liver transcription was higher for genes related to inflammation, apoptosis and cell mediated cytotoxicity (CMC). Furthermore, comparison of the differential response of S. senegalensis genes in vaccinated and unvaccinated fish to Phdp infection allowed the identification of a potential biosignature, consisting in 10 genes, as a surrogate of protection and therefore, as indicator of vaccine success against fotobacteriosis after IP vaccination. These results provide important insights into the S. senegalensis protection against Phdp induced by vaccination.

Use of in vivo induced technology to identify antigens expressed by Photobacterium damselae subsp. piscicida during infection of Senegalese sole (Solea senegalensis).

Photobacterium damselae subsp. piscicida (Phdp), the causative agent of photobacteriosis, is an important pathogen in marine aquaculture that affects many different fish species worldwide, including Solea senegalensis, an important fish species for aquaculture in the south of Europe. Bacteria express different repertoires of proteins in response to environmental conditions and when invading a host, sense in vivo environment and adapt by changing the expression of specific proteins. In the case of pathogens, identification of genes with up-regulated expression in vivo compared to in vitro conditions might give an insight into the genes relevant to the bacterial virulence. In the present work, in vivo induced antigen technology (IVIAT) has been used to search for Phdp genes only expressed or up-regulated in infected S. senegalensis. An expression library from Phdp was assayed against pooled sera from convalescent S. senegalensis specimens and 18 clones were positive, indicating that proteins encoded are expressed by Phdp during S. senegalensis infection and are immunogenic for this fish species. In addition, five proteins were reactive against adsorbed sera, indicating their in vivo induced character. Inosine-5'-monophosphate dehydrogenase, serine hydroxy methyltransferase and alanyl-tRNA synthethase, involved in aminoacid and nucleotide metabolism, the protein with antioxidant activity alkyl hydroperoxide reductase and a non-ribosomal peptide synthetase responsible for the synthesis of the siderophore piscibactin have been identified as antigens induced in Phdp during S. senegalensis infection. Proteins induced during in vivo growth of Phdp represent promising targets for the development of novel antimicrobial or prophylactic agents in the treatment and prevention of photobacteriosis.

Dietary administration of sodium alginate ameliorated stress and promoted immune resistance of grouper Epinephelus coioides under cold stress.

Grouper, Epinephelus coioides, fed a diet containing sodium alginate at 0 (control, named C) and 1.0 g kg-1 (named S) at a temperature of 28 °C for 12 days, were then further individually transferred to 28 (two groups named C-28 and S-28) or 20 °C (two groups named C-20 and S-20), and immune parameters and stress indexes were measured at the beginning and after 6, 12, 24 and 48 h of exposure. Examination of immune parameters revealed that the alternative complement activity (ACH50), lysozyme activity, phagocytic activity, superoxide dismutase, and respiratory bursts significantly increased in groupers fed the sodium alginate-containing diet for 12 days, and were higher in the S-28 than those of the C-28 and S-20 groups, which were higher than those of the C-20 group from 6 to 48 h except for ACH50 at 48 h, respiratory bursts at 48 h, and lysozymes at 6 h. For the assessment of stress indicators, cortisol, glucose, and lactate levels of serum significantly decreased in grouper fed the sodium alginate-containing diet for 12 days, and were higher in the C-20 group than those of the C-28 and S-20 groups, which were higher than those of the S-20 group at 6-48 h. In another experiment, grouper fed the test diet for 12 days at a temperature of 28 °C were challenged with Photobacterium damselae subsp. piscicida at a dose of 5 × 103 colony-forming units (cfu) (g fish)-1, and then individually transferred to 28 or 20 °C. The survival rate of challenged fish of the C-28 group was significantly lower than those of challenged fish of the C-20 and S-28 groups, which were significantly lower than that of challenged fish of the S-20 group. All challenged fish of the S-20 group survived. Survival rates over 144 h were 30.0%, 70.0%, and 56.7% for the C-28, C-20, and S-28 groups, respectively. Our results indicated that dietary sodium alginate administration downregulated stress response indicators, enhanced immune responses, and prevented impacts of physiologic stress responses, immunosuppression, and susceptibility to P. damselae subsp. piscicida in grouper subjected to cold stress. Grouper cultured at 28 °C were more susceptible to P. damselae subsp. piscicida infection.

Comparative proteome analysis reveals proteins involved in salt adaptation in Photobacterium damselae subsp. piscicida.

Proteomic approaches were applied to investigate whether Photobacterium damselae subsp. piscicida (Phdp) can directly sense and respond to growth conditions under different salinities, 0.85% and 3.5% NaCl concentrations, mimicking the osmotic conditions in host and marine water bodies, respectively. Proteins significantly altered were analyzed by two-dimensional gel electrophoresis (2-DE), liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-Q-TOF MS/MS) and bioinformatics analysis, thus resulting in 16 outer membrane proteins (OMPs), 12 inner membrane proteins (IMPs), and 20 cytoplasmic proteins (CPs). Quantitative real-time PCR was also applied to monitor the mRNA expression level of these target proteins. Cluster of orthologous groups of protein (COG) analysis revealed that when shifting from 3.5% to 0.85% salinity, the majority of the up-regulated proteins were involved in posttranslational modification, protein turnover, and chaperones, while the down-regulated proteins were mainly related to energy production and conversion, compatible solutes (carbohydrates, amino acids and their derivatives) biogenesis and transport. Differentially expressed proteins identified in the current study could be used to elucidate the salt adaptation mechanisms of Phdp during their transition between host cells and the marine habitats.

Dietary tryptophan and methionine as modulators of European seabass (Dicentrarchus labrax) immune status and inflammatory response.

Amino acids regulate key metabolic pathways important to immune responses and their nutritional supply may increase synthesis of immune-related proteins. The present study aimed to evaluate the effects of dietary supplementation of tryptophan and methionine on European seabass (Dicentrarchus labrax) cellular and humoral status. The immunomodulatory effects of tryptophan and methionine during an inflammatory insult was also evaluated after intraperitoneal injection with inactivated Photobacterium damselae subsp. piscicida (Phdp). A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). Two other diets were formulated similar to the control but including L-tryptophan or L-methionine at ×2 the requirement level (diets TRP and MET, respectively). European seabass weighing 275 g were fed the experimental diets for a period of 15 days before being sampled (trial 1). Then, fish were subjected to a peritoneal inflammation by intraperitoneally injecting UV killed Phdp (10(6) colony forming units ml(-1)) and sampled following 4 and 24 h post-injection (trial 2). Fish injected with a saline solution served as control. The haematological profile, peripheral cell dynamics and several plasma immune parameters were determined in trials 1 and 2, whereas cell migration to the inflammatory focus was also determined in trial 2. MET positively affected European seabass immune status by improving the peripheral leucocyte response, complement activity and bactericidal capacity, a stronger cellular recruitment to the inflammatory focus, and higher plasma peroxidase and bactericidal activities. TRP also seemed to improve immunostimulation, as there was a trend to augment both cell-mediated immunity and humoral capacity. However, TRP failed to improve an inflammatory response, verified by a decrease in blood phagocyte numbers and lack of immune cells recruitment. In summary, it is confirmed that MET has a pronounced influence on the innate immune response to inflammation, which is more evident than TRP, and raises its potential to incorporate in functional feeds to be used in prophylactic strategies against predictable unfavourable events.

Immunization of sea bream (Sparus aurata) juveniles against Photobacterium damselae subsp. piscicida by short bath: Effect on some pro-inflammatory molecules and the Mx gene expression.

Cytokines are a family of proteins derived from macrophages, lymphocytes, granulocytes, mast cells and epithelial cells and can be divided into interferons (IFNs), Interleukins (ILs) and Tumor Necrosis factors (TNFs) among others. The presence of cytokines in a wide number of fish species has been proved and several molecules types have been already cloned and sequenced. In this work some proinflamatory molecules and Mx gene were detected in the liver of vaccinated sea bream juveniles with an average body weight of 5 g. The method of immunization was by short bath and three different bacterins against the marine pathogen Photobacterium damselae subsp. piscicida were designed and used to immunize fish. Five genes encoding for five different molecules were analyzed by real time PCR: IL-1ß, IL Ir-2, Cox-2, Mx and TNFα. Gene expression was quantified along four days after fish immunization and results were compared among groups. Results show that the heat-inactivated vaccine stimulates the up-regulation of IL-1ß, IL Ir-2, Cox-2 and TNFα genes whereas the UV-light inactivated vaccine was the unique vaccine which stimulates the expression of Mx gene. The present is a novel study that shows by the first time the effect of the inactivation process of vaccines on the expression levels of genes involved in the defense against Photobacterium damselae subsp piscicida.