Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis.

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5'-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.

The moonlighting protein fructose 1,6-bisphosphate aldolase as a potential vaccine candidate against Photobacterium damselae subsp. piscicida in Asian sea bass (Lates calcarifer).

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.

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.

Dietary Methionine Improves the European Seabass (Dicentrarchus labrax) Immune Status, Inflammatory Response, and Disease Resistance.

Methionine presents a pivotal role in the regulation of many cellular events with crucial impact on the immune system, such as in processes involved in the control of inflammation and polyamines synthesis. Accordingly, the present study aimed to assess the modulatory effects of dietary methionine on the European seabass (Dicentrarchus labrax) immune status, inflammatory response and disease resistance to Photobacterium damselae subsp. piscicida (Phdp). For this purpose, fish were randomly distributed in three independent groups (three replicates per group) and each was fed the corresponding diet: a control diet (CTRL) formulated to meet the established amino acid requirements for the species; a diet supplemented with methionine at 0.5% of feed weight relative to the CTRL diet (8.2% of methionine concentration above CTRL); and one supplemented with methionine at 1% of feed weight to the CTRL diet (11.8% of methionine concentration above CTRL). To evaluate the immune status of fish fed with each of the diets before being submitted to bacterial infection fish were sampled from each group at 2 and 4 weeks after the beginning of feeding. Non-sampled fish were injected intraperitoneally with Phdp (5 × 103 cfu/fish) at 4 weeks after initiation of feeding and the inflammatory response (at 4, 24, and 48 h post-infection) and survival (lasting 21 days post-infection) evaluated. Fish hematological profile, peripheral cell dynamics, plasma humoral immune parameters, leucocyte migration to the inflammatory focus and head-kidney gene expression were evaluated. Results show that methionine dietary supplementation improves seabass cellular immune status without evidence of activation of pro-inflammatory mechanisms. Additionally, the observed enhanced immune status provided by methionine supplementation translated into an improved immune response to infection, as higher cellular differentiation/proliferation and recruitment to the inflammatory focus, improved plasma humoral immune parameters and modulation of key immune-related genes was observed. Lastly, after a bacterial challenge, higher survival was observed in fish fed supplemented diets, ultimately corroborating the positive effect of methionine administration for 4 weeks in the cellular immune status.

Local immune response of two mucosal surfaces of the European seabass, Dicentrarchus labrax, fed tryptophan- or methionine-supplemented diets.

Immune responses relies on an adequate provision of multiple nutrients that sustain the synthesis of key effector molecules. These needs are depicted in the already reported increase of circulating free amino acids in fish under stressful conditions. Since aquaculture and the inherent fish welfare are an emergent call, the immunomodulatory effects of amino acids on gut- and skin-associated lymphoid tissues of the European seabass (Dicentrarchus labrax) were studied under unstressed conditions and after an inflammatory insult. To achieve this goal, fish were distributed in duplicate tanks (fifteen fish per tank) and were fed for 14 days with methionine or tryptophan-supplemented diets at 2× dietary requirement level (MET and TRP, respectively) or a control diet meeting the amino acids requirement levels (CTRL). Afterwards, samples of skin and posterior gut were collected from 6 fish per dietary treatment for the assessment of the immune status while the remaining animals were intraperitoneally-injected with inactivated Photobacterium damselae subsp. piscicida and subsequently sampled either 4 or 24 h post-injection. The immune status of both mucosal surfaces was poorly affected, although a tryptophan effect was denoted after bacterial inoculation, with several immune-related genes up-regulated in the gut at 4 h post-injection, which seems to suggest a neuroendocrine-immune systems interaction. In contrast, skin mucosal immunity was inhibited by tryptophan dietary supplementation. Regarding methionine, results were often statistically non-significant, though increasing trends were denoted in a few parameters. Overall, dietary methionine did not significantly affect neither gut nor skin immunity, whereas tryptophan supplementation seems to induce modulatory mechanisms that might be tissue-specific.

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.

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.

Molecular characterization and functional analysis of a peroxiredoxin 1 cDNA from golden pompano (Trachinotus ovatus).

Peroxiredoxin 1 (Prx 1) is an important antioxidant protein that can protect organisms against the toxicity of reactive oxygen species. In this study, a full-length Prx 1 cDNA sequence (ToPrx 1) was identified from golden pompano (Trachinotus ovatus). The ToPrx 1 cDNA was 1049 base pairs (bp) long and contained a 5'-untranslated region (UTR) of 127 nucleotides, a 3'-UTR of 328 nucleotides, and a 594 bp open reading frame (ORF) encoding a 197 amino acid polypeptide. The ToPrx 1 protein showed strong homology (79-91%) with Prx 1 proteins from other species and contained the conserved Prx domain and the signature of the peroxidase catalytic center. Phylogenetic analysis revealed that ToPrx 1 was in the fish Prx 1 subgroup, which suggests that ToPrx 1 could belong to the 2-Cys Prx subgroup. ToPrx 1 mRNA was ubiquitously detected in all tested tissues, and its expression was comparatively high in the fin, spleen, kidney, intestine, eye, gill, and blood. The expression levels of ToPrx 1 mRNA were significantly up-regulated in liver, spleen, kidney, and intestine of golden pompano injected with Photobacterium damselae. The recombinant ToPrx 1 protein (rToPrx 1) was expressed and purified through affinity chromatography and refolded successfully using ion-exchange chromatography. The antioxidant activity assay of rToPrx 1 showed that it could reduce insulin in the presence of dithiothreitol, which suggests that the antioxidant function of rToPrx 1 is thiol dependent. This study provides useful information to help further understand the functional mechanism of Prx 1 in marine fish immunity.

Protective efficacy and immune responses induced by a DNA vaccine encoding codon-optimized PPA1 against Photobacterium damselae subsp. piscicida in Japanese flounder.

Photobacterium damselae subsp. piscicida (Pdp) kills many cultured marine fish. As it evolves resistance to existing vaccines, new vaccines are needed. PPA1 is a major antigenic protein of Pdp. Here, DNA vaccines encoding wild-type PPA1 (pPPA1(wt)) and codon-optimized PPA1 (pPPA1(opt)) were constructed and tested against Pdp in Japanese flounder. The mRNA levels of the two antigenic genes at the vaccination site were not different, but the protein level was significantly higher in the pPPA1(opt)-vaccinated fish. In addition, after a bacterial challenge, the levels of interleukin (IL)-1ß, IL-6 and IFN-γ mRNA significantly increased in the pPPA1(opt)-vaccinated fish but not in the pPPA1(wt)-vaccinated fish. The relative percent survival (RPS) after the challenge was higher in the pPPA1(opt)-vaccinated fish (90.9) than in the pPPA1(wt)-vaccinated fish (69.2). At the early stage of the infection after the challenge, the number of viable Pdp in the spleen was significantly lower in the pPPA1(opt)-vaccinated fish than in the pPPA1(wt)-vaccinated fish. These data show that codon-optimized DNA vaccine pPPA1(opt) had a strong immunogenicity and conferred protective efficacy against Pdp infection in Japanese flounder.

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1ß, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Photobacteriosis: prevention and diagnosis.

Photobacteriosis or fish pasteurellosis is a bacterial disease affecting wild and farm fish. Its etiological agent, the gram negative bacterium Photobacterium damselae subsp. piscicida, is responsible for important economic losses in cultured fish worldwide, in particular in Mediterranean countries and Japan. Efforts have been focused on gaining a better understanding of the biology of the pathogenic microorganism and its natural hosts with the aim of developing effective vaccination strategies and diagnostic tools to control the disease. Conventional vaccinology has thus far yielded unsatisfactory results, and recombinant technology has been applied to identify new antigen candidates for the development of subunit vaccines. Furthermore, molecular methods represent an improvement over classical microbiological techniques for the identification of P. damselae subsp. piscicida and the diagnosis of the disease. The complete sequencing, annotation, and analysis of the pathogen genome will provide insights into the pathogen laying the groundwork for the development of vaccines and diagnostic methods.

Evaluating the protective efficacy of antigen combinations against Photobacterium damselae ssp. piscicida infections in cobia, Rachycentron canadum L.

Cobia, Rachycentron canadum L., is a very important aquatic fish that faces the risk of infection with the bacterial pathogen Photobacterium damselae ssp. piscicida, and there are few protective approaches available that use multiple antigens. In the present study, potent bivalent antigens from P. damselae ssp. piscicida showed more efficient protection than did single antigens used in isolation. In preparations of three antigens that included recombinant heat shock protein 60 (rHSP60), recombinant α-enolase (rENOLASE) and recombinant glyceraldehyde-3-phosphate dehydrogenase (rGAPDH), we analysed the doses that elicited the best immune responses and found that this occurred at a total of 30 µg of antigen per fish. Subsequently, vaccination of fish with rHSP60, rENOLASE and rGAPDH achieved 46.9, 52 and 25% relative per cent survival (RPS), respectively. In addition, bivalent subunit vaccines--combination I (rHSP60 + rENOLASE), combination II (rENOLASE + rGAPDH) and combination III (rHSP60 + rGAPDH)--were administered and the RPS in these groups (65.6, 64.0 and 48.4%, respectively), was higher than that achieved with single-antigen administration. Finally, in combination IV, the trivalent vaccine rHSP60 + rENOLASE + rGAPDH, the RPS was 1.6%. Taken together, our results suggest that combinations of two antigens may achieve a better efficiency than monovalent or trivalent antigens, and this may provide new insights into pathogen prevention strategies.

Development of an in vitro assay based on humoral immunity for quality control of oil-adjuvant Pseudotuberculosis vaccine in Yellowtail Seriola quinqueradiata.

Photobacterium damselae subsp. piscicida is an infectious pathogen that causes Pseudotuberculosis in Yellowtail fish. In Japan, several oil-adjuvant vaccines for Pseudotuberculosis have been approved for control of infectious diseases in aquaculture. Before distribution of an approved fish vaccine, an artificial challenge test for quality control is performed by the manufacturer and National Veterinary Assay Laboratory under Pharmaceutical Law of Japan to confirm potency. In this study, artificial challenge tests with a range of five diluted or undiluted approved vaccines was performed to determine the relationship between antigen levels and vaccine efficacy. Immunization of fish with the undiluted vaccine prevented Pseudotuberculosis. Results of artificial challenge tests demonstrated vaccine efficiency was dose dependent. Agglutination assays using immune sera were performed to determine agglutination titers, which were also dose dependent. These results suggest a link between survival rate in the artificial challenge tests and agglutination titers. Western blotting analysis identified a specific protein approximately 37 kDa in size in vaccinated fish. We confirmed antibodies were produced in vaccinated fish by immunoreactions with the approved vaccine. An agglutination assay based on humoral immunoreactions would be a useful alternative to the artificial challenge test for quality control of vaccines for aquaculture.

Diet supplementation of Pediococcus pentosaceus in cobia (Rachycentron canadum) enhances growth rate, respiratory burst and resistance against photobacteriosis.

Cobia (Rachycentron canadum) is an economically important fish species for aquaculture in tropical and sub-tropical areas. Cobia aquaculture industry has severely damaged due to photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp), especially in Taiwan. Antibiotics and vaccines have been applied to control Pdp infection, but the efficacy has been inconsistent. One species of lactic acid bacteria, Pediococcus pentosaceus strain 4012 (LAB 4012), was isolated from the intestine of adult cobia, and its culture supernatant can effectively inhibit Pdp growth in vitro. The acidic pH derived from metabolic acids in LAB culture supernatant was demonstrated to be an important factor for the suppression. After a 2-week feeding of LAB 4012, the growth rate of the fed cobia was 12% higher than that of the non-fed group, and the relative percentage of survival (RPS) of the fed cobia was found to be 74.4 in Pdp immersion challenge. In addition, the respiratory burst (RB) of peripheral blood leukocytes (PBL) in the LAB 4012-fed group was significantly higher than that of the non-fed group. Although feeding LAB 4012 did not improve specific antibody response in cobia after immunization with Pdp vaccine, it still significantly raised the survival rate by 22% over that of the non-fed group after Pdp immersion challenge. Judging by the quick induction of high protection against Pdp infection and promotion of growth in larvae, LAB 4012 was considered to be a viable probiotic for cobia aquaculture.

High mortality of juvenile gilthead sea bream (Sparus aurata) from photobacteriosis is associated with alternative macrophage activation and anti-inflammatory response: results of gene expression profiling of early responses in the head kidney.

The halophilic bacterium Photobacterium damselae subsp. piscicida (Phdp) represents a substantial health problem for several fish species in aquaculture. Bacteria that reside free and inside phagocytes cause acute and chronic forms of photobacteriosis. Infections of juveniles rapidly kill up to 90-100% fish. Factors underlying failure of the immune protection against bacteria remain largely unknown. The reported study used a transcriptomic approach to address this issue. Juvenile sea breams (0.5 g) were challenged by immersion in salt water containing 2.89 × 10(8) CFU of a virulent Phdp and the head kidney was sampled after 24- and 48-h. Analyses were performed using the second version of a 44 k oligonucleotide DNA microarray that represents 19,734 sea bream unique transcripts and covers diverse immune pathways. Expression changes of selected immune genes were validated with qPCR. Results suggested rapid recognition of the pathogen, as testified by up-regulation of lectins and antibacterial proteins (bactericidal permeability-increasing protein lectins, lysozyme, intracellular and extracellular proteases), chemokines and chemokine receptors. Increased expression of proteins involved in iron and heme metabolism also could be a response against bacteria that are dependent on iron. However, negative regulators of immune/inflammatory response were preponderant among the up-regulated genes. A remarkable finding was the increased expression of IL-10 in concert with up-regulation of arginase I and II and proteins of the polyamine biosynthesis pathway that diverts the arginine flux from the production of reactive nitrogen species. Such expression changes are characteristic for alternatively activated macrophages that do not develop acute inflammatory responses. Immune suppression can be induced by the host to reduce tissue damages or by the pathogen to evade host response.

Cellular and humoral immune responses of Senegalese sole, Solea senegalensis (Kaup), following challenge with two Photobacterium damselae subsp. piscicida strains from different geographical origins.

The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate-buffered saline (control) or two concentrations (2 × 10² and 2 × 106 CFU mL⁻¹) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.

Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate.

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 µg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata L.) did not correlate with disease resistance to Photobacterium damselae.

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabream's immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.