Lyme borreliosis (LB), caused by different species belonging to the Borrelia burgdorferi sensu lato group, is the most common tick-borne disease in Europe. However, its true burden remains difficult to assess, partly due to the diagnostic variability of commercial assays. This study evaluated seroprevalence and seroconversion following tick bite, using four serological assays with distinct antigenic compositions. Participants provided blood samples within three days of the tick bite and three months after to assess seroconversion. We evaluated the seroprevalence, seroconversion, inter-assay variation, consensus scoring, and factors affecting the detection rate for both seroprevalence and seroconversion.Seroprevalence evaluation varied across the four serology assays: 33% using Anti-Borrelia plus VlsE (ABV), 26% using Epitogen™ Lyme (Epitogen), 22% using C6 Lyme ELISA (C6) and 10% using IDEIA™ B. burgdorferi (IDEIA). Estimation of overall seroconversion rates also differed: C6 had the highest rate (93%), followed by Epitogen (78%), ABV (58%) and IDEIA (38%). Using a consensus scoring approach that required positivity of at least two assays (including immunoblot) yielded a seropositivity rate of 23% at inclusion and an overall seroconversion rate of 86%. The Epitogen assay showed the highest level of concordance with the consensus score, followed by C6, ABV, and IDEIA assays.Seroprevalence and seroconversion estimates in LB are highly assay-dependent. Assay choice significantly influenced outcome, mainly reflecting differences in antigen composition. This study underscores the importance of evaluating the antigen composition used in assays to ensure accurate result interpretation, particularly in the context of potential species variability across differing geographical regions. The use of consensus scoring across complementary multi-assays, or the implementation of well-designed assays with appropriate antigen coverage, offers a pathway to improved diagnostic accuracy and enhanced comparability.

Bactericidal/permeability-increasing protein (BPI) is an antimicrobial protein showing bactericidal, opsonic and anti-inflammatory activities with therapeutic potential. However, the potential role of BPI on immunological properties in teleost is not fully understood. In this study, a BPI2-like gene was successfully cloned from American eel (Anguilla rostrata), which contains an open reading frame (ORF) of 1422 bp encoding 473 amino acids. The ArBPI2 consists of a signal peptide of 19 amino acids, an N-terminus LPS-binding domain, a C-terminal domain, and a basic pI of 9.0, typical attribute of BPI proteins found in mammals. The N-terminal domain of ArBPI2 (27-249 amino acids, ArBPI2-ND) was produced as a recombinant protein (rArBPI2-ND) and its antimicrobial activity was performed. ArBPI2-ND exhibited a strong affinity for LPS, and had a broad binding capacity for both Gram-negative and Gram-positive bacteria. The minimum inhibitory concentration (MIC) assays revealed that rArBPI2-ND exerted stronger antibacterial activity on Gram-negative bacteria than on Gram-positive bacteria. Further study revealed that rArBPI2-ND exerted its bactericidal activity against Aeromonas veronii by inhibiting the formation of bacterial biofilms, increasing the outer membrane permeability, destroying the cell membrane structure and binding to genomic DNA, leading to cytoplasmic leakage and bacterial death. This is the first report on the molecular characteristics, biological functions, and bactericidal mechanism of the N-terminal domain of a BPI protein from A. rostrata.

Nile tilapia (Oreochromis niloticus) has become one of the most economically valuable fish species in global aquaculture. Recently, the industry has been increasingly affected by bacterial infections, particularly Streptococcus agalactiae, which has become a dominant pathogen causing severe disease outbreaks and economic losses. Interleukin (IL)-11 is a versatile cytokine involved in immune modulation, but its adjuvant potential has been rarely studied in fish. In this study, recombinant Nile tilapia IL-11 (rIL-11) was evaluated as a molecular adjuvant by co-injecting it with a formalin-killed whole-cell (FKC) S. agalactiae vaccine. Fish were divided into three groups: PBS (control), FKC alone, and FKC + rIL-11. Co-injection with rIL-11 significantly enhanced both innate and adaptive immune responses. Fish receiving FKC + rIL-11 showed significantly higher lysozyme, myeloperoxidase, glutathione peroxidase, and glutathione reductase activity compared to controls. Moreover, significantly increased IgM antibody levels were detected at all sampling points post-vaccination in the FKC + rIL-11 group, together with higher densities of IgM+ B cells in the spleen. Importantly, cytokine gene expression analysis revealed significant upregulation of TNF-α, IL-6, IL-8, IL-1β, IL-11a, and IL-11b in the spleen, and TNF-α and IL-8 in the liver, particularly during the early phase (days 1-3). Protective efficacy testing showed that the FKC + rIL-11 group achieved a relative percent survival (RPS) of 78.78 %, significantly higher than the 60.60 % observed with FKC alone. Overall, these results demonstrate that rIL-11 serves as a potent vaccine adjuvant, enhancing both humoral and cellular responses, and improving protection of Nile tilapia against S. agalactiae.

Aquaculture is a central commercial commodity for low-to middle-income countries and a promising source of economically important food and livelihood. Indian aquaculture faces challenges with the onset of several upcoming fish diseases. The antimicrobial resistance (AMR) pathogen Edwardsiella tarda infection is one of the serious threats affecting worldwide aquaculture. Replacing antibiotics, vaccines can be used as a safe treatment for multidrug-resistant E. tarda. The present study focuses on creating a formalin killed vaccine incorporated with IFN-γ and β-glucan as adjuvants, that confers protection against E. tarda infection in Nile tilapia (Oreochromis niloticus). The isolated virulent strain was cultivated in marine nutrient broth and inactivated by 0.5 % formalin treatment. To increase the immunogenicity, the bacterial suspension was mixed with IFN-γ and β-glucan. Fish were intraperitoneally immunized with IFN-γ and β-glucan -adjuvanted formalin killed vaccine (FKV), followed by sampling at a fixed time. After 28 days from the first immunization, the fish were challenged with E. tarda at a dose of 1 x 107 CFU/fish. Blood from the immunized fish was sampled weekly to measure immunochemical parameters, including sodium oxide dismutase (SOD), myeloperoxidase (MPO), lysozyme activity (LZM), and bactericidal activity. Fish serum IgM titre values were measured on the 14th and 28 day post-vaccination (d.p.v.), and both showed that the adjuvanted vaccinated groups had higher antibody levels than the control group. The vaccinated groups with IFN-γ and β-glucan showed considerably higher expressions immune genes, including IgM, TCR β, MHC II. The study emphasizes that β-glucan and IFN-γ adjuvanted inactivated vaccine is a potentially plausible approach toward novel vaccination strategies for sustainable aquaculture.

Colony-stimulating factor-1 (CSF-1) receptor, also known as macrophage colony-stimulating factor (MCSF) receptor, belongs to the type III protein tyrosine kinase receptor family. MCSF and IL-34 play essential roles in both innate and adaptive immune systems in vertebrates through their shared receptor MCSFR. While the functional study of MCSFR in mammals has been well-demonstrated, its role in fish remains limited. Therefore, this report aims to identify and study the expression of the MCSFR genes in rainbow trout Oncorhynchus mykiss, where four paralogues were found present at different genomic loci, two identified for the first time in this study. The deduced protein structure of these MCSFRs reveals five immunoglobulin (Ig)-like domains, a transmembrane domain and a conserved intracellular domain containing a glycine-rich motif (Gly-x-Gly-x-x-Gly), similar to other species. Phylogenetic and synteny analyses demonstrate that MCSFR are present throughout vertebrates, with two forms present in teleost fish more generally (type I and type II MCSFR), existing as pairs of genes (MCSFR1a/ MCSFR1b, MCSFR2a/MCSFR2b) in trout. The MCSFR genes are widely expressed, with higher transcript levels observed in immune tissues such as the spleen, blood and head kidney. The paralogues showed marked differences in expression modulation. Following Yersinia ruckeri infection, MCSFR2a was highly induced but after stimulation of RTS-11 cells, a trout monocyte/macrophage-like cell line, with Y. ruckeri flagellin both MCSFR1b and MCSFR2a were induced. However, none of the different paralogues of MCSFR were induced by proinflammatory cytokines (trout rTNF-alpha, rIL-6 and rIFN-gamma). This study adds to our knowledge of the molecules/ pathways present in fish that drive macrophage regulation and activation, and emphasizes the complexity present with multiple ligands and receptors involved.

Aquaculture industry frequently encounters disease outbreaks, high mortalities, as well as emergence of new pathogens due to its intensification. Streptococcus agalactiae (Lancefield’s group B Streptococcus) is an important pathogen extensively causing infectious disease in tilapia resulting in huge economic loss and mortality. To date, vaccination has proved to be successful in defending against infectious diseases prevailing among farmed fish species. This study aimed to develop an S. agalactiae inactivated vaccine (SAIV) using molecular adjuvants, flagellin and tilapia interferon gamma (IFN-γ), and to assess the generated immune response and protective efficacy of the adjuvant incorporated vaccine against S. agalactiae infection in Nile tilapia. The fish were vaccinated with SAIV together with either flagellin or IFN-γ and both together by intraperitoneal injection. The vaccinated fish were challenged with a virulent strain of S. agalactiae on day 36 and monitored for 3 weeks to assess cumulative mortality. The results showed that the vaccine offered significant protection with relative percentage survival (RPS) of 59.37

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDETM GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDETM GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDETM GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feedbased adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDETM GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

Lyme Borreliosis (LB), or Lyme disease, is a growing health concern caused by Borrelia burgdorferi sensu lato (Bbsl) bacteria transmitted through tick bites, and untreated cases can lead to severe health complications. Existing serology tests, while valuable, have low sensitivity in early infection stages where diagnosis is vital, interpretation variability, and false positives from cross-reactivity, while direct detection methods also suffer from low sensitivity, due to the inconsistent presence of Bbsl components in clinical samples. This study validated the diagnostic performance of the novel Epitogen Lyme Detect IgG enzyme-linked immunosorbent assay (ELISA) based on scaffold-displayed peptide antigens, using 120 specific immunodominant epitopes selected from 37 antigenic bacterial proteins corresponding to the main pathogenic Bbsl genospecies. Using 220 serum samples from Scottish patients with early, late, and disseminated LB, the assay's sensitivity was compared with that of the LIAISON Borrelia IgG CLIA, while specificity was assessed with 198 control samples, including healthy individuals and patients with diseases that are humorally similar. The Epitogen Lyme Detect IgG assay demonstrated comparable performance to the LIAISON Borrelia IgG in disseminated and late LB (Lyme neuroborreliosis, acrodermatitis chronica atrophicans, and Lyme arthritis). Notably, the Epitogen Lyme Detect IgG showed significantly higher sensitivity in patients with suspected erythema migrans, while maintaining high specificity. The Epitogen Lyme Detect IgG ELISA offers a promising advancement in LB diagnostics, demonstrating its potential for more accurate and timely diagnosis, particularly in the early stages of LB infection.IMPORTANCELyme Borreliosis (LB), caused by Borrelia burgdorferi sensu lato bacteria, poses significant health risks if undiagnosed or diagnosed late. Current diagnostic tests have limitations, especially in early-stage detection. This study validates the Epitogen Lyme Detect IgG enzyme-linked immunosorbent assay, demonstrating superior sensitivity in early LB detection while maintaining high specificity. The Epitogen Lyme Detect IgG comprises a suite of 120 immunodominant IgG epitopes/peptides from 37 bacterial antigens, covering the main LB-causing species: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, and Borrelia mayonii. The novel design of multiplexing peptide antigens onto a scaffold to facilitate expression, correct folding, and orientation of the relevant peptides offers a promising advancement, potentially leading to more accurate and timely LB diagnoses and improving patient outcomes.

Streptococcus agalactiae is regarded as a major bacterial pathogen of farmed fish, with outbreaks in Nile tilapia causing significant losses. Vaccination is considered the most suitable method for disease control in aquaculture, with the potential to prevent such outbreaks if highly efficacious vaccines are available for use. Several vaccines have been produced to protect against S. agalactiae infection in tilapia, including inactivated vaccines, live attenuated vaccines, and subunit vaccines, with variable levels of protection seen. Two commercial adjuvants, Montanide™ ISA 763A VG and ISA 763B VG, have been developed recently and designed to improve the safety and efficacy of oil-based emulsions delivered by intraperitoneal injection. In particular, their mode of action may help identify and stimulate particular immunological pathways linked to the intended protective response, which is an important tool for future vaccine development. Therefore, this study aimed to characterize the potential of two adjuvanted-bacterial vaccines against S. agalactiae (SAIV) comparatively, to determine their usefulness for improving protection and to analyse the immune mechanisms involved. Nile tilapia were divided into four groups: 1) fish injected with PBS as a control, 2) fish injected with the SAIV alone, 3) fish injected with the SAIV + Montanide™ ISA 763A VG, and 4) fish injected with the SAIV + Montanide™ ISA 763B VG. Following immunization selected innate immune parameters were analysed, including serum lysozyme, myeloperoxidase, and bactericidal activity, with significantly increased levels seen after immunization. Cytokines associated with innate and adaptive immunity were also studied, with expression levels of several genes showing significant up-regulation, indicating good induction of cell-mediated immune responses. Additionally, the specific IgM antibody response against S. agalactiae was determined and found to be significantly induced post-vaccination, with higher levels seen in the presence of the adjuvants. In comparison to the protection seen with the unadjuvanted vaccine (61.29% RPS), both Montanide™ ISA 763A VG and Montanide™ ISA 763B VG improved the RPS, to 77.42% and 74.19% respectively. In conclusion, Montanide™ ISA 763A VG and Montanide™ ISA 763B VG have shown potential for use as adjuvants for fish vaccines against streptococcosis, as evidenced by the enhanced immunoprotection seen when given in combination with the SAIV vaccine employed in this study.

Dietary supplements containing a functional feed additive have been shown to be beneficial to fish and shellfish aquaculture. However, the functional properties of aquafeed formulations have rarely been reported in fish. This study aimed to investigate the effects of natural free amino acid mix (FAAM) supplementation as a functional solution on the growth performance and nutrient utilization in a carnivorous fish, Asian seabass (Lates calcarifer). Five isonitrogenous and isolipidic diets were prepared with graded supplementation levels of FAAM at 0 % (control group), 0.25 %, 0.50 %, 0.75 %, and 1.0 %, denoted as FAAM0, FAAM0.25, FAAM0.5, FAAM0.75, and FAAM1.0, respectively. The experimental fish were fed different dietary FAAM supplementations to apparent satiation twice daily for eight weeks. Significant improvements were observed in the growth performance of fish among the five groups (P < 0.05). Fish fed with FAAM0.75 displayed significantly increased activities of lysozyme, myeloperoxidase, catalase, and glutathione peroxidase (P < 0.05). The activities of digestive enzymes, including amylase, protease, and lipase, were enhanced by the supplementation of FAAM in the feed (P < 0.05), especially for the groups that contained more than 0.5 % FAAM in the feed. Furthermore, the morphological profile of the intestinal tract, including the mucosal fold height, width, thickness, and goblet cell, increased in fish fed with FAAM at 1.0 % (P < 0.05). Moreover, FAAM supplementation in diets not only modulated the expression of immune-related genes (glutathione peroxidase (GPx), complement (C)3, C4, and C-reactive protein) in the liver but also positively impacted the growth-ralated genes, including growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I), and IGF-II. In addition, the amounts of monounsaturated fatty acids (mainly oleic acid (C18:1n9c)) and polyunsaturated fatty acids-especially γ-linolenic acid (C18:3 n6) and α-linolenic acid (C18:3n3)-increased in fish fed with diets containing FAAMs (P < 0.05). Interestingly, the diets supplemented with FAAMs also had a positive effect on the economic indices in terms of revenue-to-cost ratios. These findings provide a scientific basis for the application of FAAMs as a functional solution that can be used in feed formulations for Asian seabass.

Serum amyloid A (SAA) proteins belong to a family of acute-phase reactants, playing an integral role in defending the organism from pathological damage. Despite a wealth of data on the regulation of SAA transcripts in teleosts, there is only limited information on these proteins' abundance in fish. The aim of this study is to characterise SAA protein levels in salmonids using a newly developed antibody specific to salmonid SAA. The salmonid SAA antibody detected SAA and accurately discriminated between stimulated and control specimens from rainbow trout macrophage cell line (RTS-11) in vitro, as well as rainbow trout challenged with Aeromonas salmonicida- or flagellin-stimulated Atlantic salmon in vivo. The presence of SAA protein was analysed in RTS-11 cell line supernatants, liver, and spleen samples using ELISA, immunoblotting, and immunohistochemistry. This study is the first to characterise SAA protein levels in salmonids in vivo and in vitro. The newly developed salmonid SAA antibody was able to discriminate between stimulated and unstimulated specimens, showing that it can be used to study the acute-phase response in salmonids with the potential to be further developed into assays to monitor and evaluate health in wild and farmed fish.

Cytokines are small proteins that regulate innate and adaptive immune responses and are released by both immune and non-immune cell types. In the current study, the constitutive and induced gene expression profiles of a suite of proinflammatory and regulatory cytokines was examined comparatively in eight rainbow trout (Oncorhynchus mykiss) cell lines, in order to establish the cytokine repertoires of these different cell types, especially the understudied non-immune cells. They included three epithelial cell lines (RTgut, RTgill, and RTL), one endothelial cell line (RTH), one fibroblast cell line (RTG-2), two stromal cell lines (TSS and TPS-2) and one monocyte/macrophage-like cell line (RTS-11). Three types of primary leukocytes (derived from blood, spleen and head kidney) of trout were also included in the analysis, to allow comparison to the repertoires expressed in T cells, as a major source of cytokines in immune responses. The major findings are: 1) IL-2A, IL-2B, IL-4/13B1, IL-4/13B2, IL-10b, P40B1, P28B, IL-17A/F1b, TNF-α3, TNF-α4, IFNγ1, CCL20L2b and CCL20L3a are expressed mainly in leukocytes but IL-17 N, IL-17D, IL-20 and CCL20L1b2 are not expressed in these cells. Hence future studies in these cell lines will help establish their function in fish; 2) Some of the cytokines were differentially expressed in the cell lines, revealing the potential role of these cell types in aspects of trout mucosal and inflammatory immune responses, 3) Similar cell types grouped together in the cell cluster analysis, including the leukocyte cluster, stromal cell cluster, and epithelial and endothelial cell cluster. Taken together, this investigation of these trout cell lines forms a good database for studying the function of cytokines not expressed in isolated leukocytes or that are preferentially expressed in the cell lines. Furthermore, the cytokine expression analysis undertaken confirmed the phenotypic relationship of these cell types at the molecular level.

AbstractColorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer‐related death. Although clinicopathological parameters provide invaluable prognostic information, the accuracy of prognosis can be improved by using molecular biomarker signatures. Using a large dataset of immunohistochemistry‐based biomarkers (n = 66), this study has developed an effective methodology for identifying optimal biomarker combinations as a prognostic tool. Biomarkers were screened and assigned to related subsets before being analysed using an iterative algorithm customised for evaluating combinatorial interactions between biomarkers based on their combined statistical power. A signature consisting of six biomarkers was identified as the best combination in terms of prognostic power. The combination of biomarkers (STAT1, UCP1, p‐cofilin, LIMK2, FOXP3, and ICOS) was significantly associated with overall survival when computed as a linear variable (χ2 = 53.183, p < 0.001) and as a cluster variable (χ2 = 67.625, p < 0.001). This signature was also significantly independent of age, extramural vascular invasion, tumour stage, and lymph node metastasis (Wald = 32.898, p < 0.001). Assessment of the results in an external cohort showed that the signature was significantly associated with prognosis (χ2 = 14.217, p = 0.007). This study developed and optimised an innovative discovery approach which could be adapted for the discovery of biomarkers and molecular interactions in a range of biological and clinical studies. Furthermore, this study identified a protein signature that can be utilised as an independent prognostic method and for potential therapeutic interventions.

Disease prevention by vaccination is, on economic, environmental and ethical grounds the most appropriate method for pathogen control currently available to the aquaculture sector. However, vaccine administration in aquatic animals faces obvious technical problems not encountered in other land animals. Thus, oral vaccines are highly demanded by the aquaculture sector that requests alternatives to the labor-intensive injectable vaccines that require individual handling of fish, provoking stress-related immunosuppression and handling mortalities. Despite this, most previous attempts to obtain effective oral vaccines have failed both in fish and mammals. This could be a consequence of very restricted tolerance mechanisms in the intestine given the fact that this mucosa is at the frontline upon antigen encounter and has to balance the delicate equilibrium between tolerance and immunity in a microbe rich aquatic environment. In this context, the search for an optimal combination of antigen and adjuvant that can trigger an adequate immune response able to circumvent intestinal tolerance is needed for each pathogen. To this aim, we have explored potential of molecules such as β-glucans, flagellin, CpG and bacterial lipopolysacharide (LPS) as oral adjuvants. For this, we have determined the effects of these adjuvants ex vivo in rainbow trout intestine tissue sections, and in vitro in leucocytes isolated from rainbow trout spleen and intestine. The effects were evaluated by analyzing the levels of transcription of different genes related to the innate and adaptive immune response, as well as evaluating the number of IgM-secreting cells. LPS seems to be the molecule with stronger immunostimulatory potential, and could safely be used as a mucosal adjuvant in rainbow trout. Moreover, the designed strategy provides a fast methodology to screen adjuvants that are suitable for oral vaccination, providing us with valuable information about how the intestinal mucosa is regulated in fish.

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-β1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-β1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

IFN-gamma is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-gamma orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-gamma protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-gamma peptides were developed and characterised to monitor IFN-gamma expression. The results show that the IFN-gamma mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-gamma protein and rainbow trout IFN-gamma produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-gamma at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-gamma secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-gamma(+) cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-gamma(+) cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-gamma in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-gamma producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Adjuvants are the helper substances that increase vaccine efficacy by enhancing the potency and longevity of specific immune responses to antigens. Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. The characterization of their mode of action is a valuable aid to future vaccine development, particularly for the potential identification and stimulation of specific immunological pathways related to the desired protective response. This study characterized the expression of selected immune-related genes in the peritoneal cavity, head kidney and spleen following the administration of two adjuvanted-bacterial vaccines thought to induce humoral (Montanide™ ISA 763A VG) or humoral and cell mediated (Montanide™ ISA 761 VG) immune responses, to determine if differences in responsiveness are readily apparent. The most informative site was the spleen, where Montanide™ ISA 763A VG + bacterin gave rise to upregulation of genes driving T-cell/lymphoid responses, namely IL-2, IL-15 and IL-21. This combined with upregulation of IFNγ1 and IFNγ2, IL-4/13B2, p35A1 and p40 (B1 and C) indicated that the induction of Th1 and possibly Th2 immunity was occurring in fish vaccinated with this adjuvant. Perhaps the most intriguing finding was the lack of a detectable Th1 response in fish given Montanide™ ISA 761 VG + bacterin, suggesting some other arm of the immune system is activated to give protection. Whatever the reason for the different responses detected, it is clear from the present study that the adjuvant used has a major impact on the responses elicited. Since these differences are readily detectable it allows, in principle, their use to help select the most appropriate adjuvants for inclusion into fish vaccines, where the type of response elicited may need to be tailored to a particular pathogen to confer protection.

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, MontanideTM ISA 763B VG and MontanideTM GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+MontanideTM ISA 763B VG; and 4) fish injected with SAGV+MontanideTM GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCR beta, IL-1 beta, IL-8 and TNF alpha) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, MontanideTM ISA 763B VG and MontanideTM GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.