Head Kidney Leukocytes Research Articles

Molecular characterization and role in virus infection of Beclin-1 in large yellow croaker (Larimichthys crocea)

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66–98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.

Identification and functional characterization of a fish-specific tlr19 in common carp (Cyprinus carpio L.) that recruits TRIF as an adaptor and induces ifn expression during the immune response

Toll-like receptor 19 (Tlr19) is a fish-specific TLR that plays a critical role in innate immunity. In the present study, we aimed to identify tlr19 from common carp (Cyprinus carpio L.) and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr19 cDNA sequence (Cctlr19) was identified in common carp. Phylogenetic analysis revealed that CcTlr19 was most closely related to Danio rerio Tlr19. Subcellular localization analysis indicates that CcTlr19 was synthesized in the free ribosome and then transported to early endosomes. Cctlr19 was constitutively expressed in all the examined tissues, with the highest expression in the brain. After poly(I:C) and Aeromonas hydrophila injection, the expression of Cctlr19 was significantly upregulated in immune-related organs. In addition, the expression of Cctlr19 was upregulated in head kidney leukocytes (HKL) upon stimulation with different ligands. Immunofluorescence and luciferase analyses indicate that CcTlr19 recruited TRIF as an adaptor. Furthermore, CcTlr19 can activate the expression of ifn-1 and viperin. Taken together, these findings lay the foundation for future research to investigate the mechanisms underlying fish tlr19.

Genomic Analysis of Pasteurella atlantica Provides Insight on Its Virulence Factors and Phylogeny and Highlights the Potential of Reverse Vaccinology in Aquaculture.

Pasteurellosis in farmed lumpsuckers, Cyclopterus lumpus, has emerged as a serious disease in Norwegian aquaculture in recent years. Genomic characterization of the causative agent is essential in understanding the biology of the bacteria involved and in devising an efficient preventive strategy. The genomes of two clinical Pasteurella atlantica isolates were sequenced (≈2.3 Mbp), and phylogenetic analysis confirmed their position as a novel species within the Pasteurellaceae. In silico analyses revealed 11 genomic islands and 5 prophages, highlighting the potential of mobile elements as driving forces in the evolution of this species. The previously documented pathogenicity of P. atlantica is strongly supported by the current study, and 17 target genes were recognized as putative primary drivers of pathogenicity. The expression level of a predicted vaccine target, an uncharacterized adhesin protein, was significantly increased in both broth culture and following the exposure of P. atlantica to lumpsucker head kidney leucocytes. Based on in silico and functional analyses, the strongest gene target candidates will be prioritized in future vaccine development efforts to prevent future pasteurellosis outbreaks.

Effects of subcutaneous injection of λ/κ-carrageenin on the immune and liver antioxidant status of gilthead seabream (Sparus aurata).

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparusaurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrioanguillarum and Photobacteriumdamselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1β and prostaglandin D synthase 1 were upregulated at 1.5 and 3hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Functional characterization of an interleukin 20 like homologue in grass carp Ctenopharyngodon idella

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and plays an important biological role in tissue homeostasis and regulation of host immune defenses. IL-20 homologues have recently been discovered in fish, but their functions have not been studied. In this study, an IL-20 like (IL-20L) cytokine was cloned in grass carp (Ctenopharyngodon idella) and its bioactivities were investigated. Expression analysis showed that the CiIL-20L gene was constitutively expressed in tissues with the highest expression detected in the head kidney. It was upregulated in the head kidney after infection with Flavobactrium columnare (F. cloumnare) and grass carp reovirus II (GCRV II). The recombinant CiIL-20L produced in E. coli cells was shown to be effective in inducing the expression of Th cytokine genes (IFN-γ, IL-4/13A, IL-4/13B and IL-10), macrophage marker genes (arginase 2, IRF4, KLF4 and SOCS3) and inflammatory genes (IL-1β, IL-6, IL-8 and TNFα) in the head kidney leukocytes when stimulated at 12 h. Long term culture (6 days) of head kidney macrophages in the presence of CiIL-20L leads to high expression of IRF4, TGFβ1 and arginase 2. Our data suggest that IL-20 may play regulatory roles in promoting Th responses, macrophage differentiation and inflammation.

In vitro effects ofAsparagus racemosusethanolic root extract on cellular immune response and immune‐related gene expression ofLabeo rohita(Hamilton, 1822) leucocytes and anti‐Aeromonas hydrophilaactivity

Asparagus racemosus is an important herb, the medicinal properties of which have been reported in traditional systems of medicine. The potential application of this plant as an immunostimulant for fish was explored in this study, using in vitro assay methods. Based on the extraction yield, phytochemical properties and antioxidant potentials, the ethanolic root extract of the plant (AREE) was selected and subsequently used to observe in vitro cellular immune responses, immune-related gene expression of Labeo rohita head kidney (HK) leucocytes, and anti-Aeromonas hydrophila activity. Different cellular immunological parameters (nitric oxide production, superoxide anion production and myeloperoxidase activity) of the HK leucocytes showed significantly enhanced responses when incubated with different concentration of AREE (6.25, 12.5, 25, 50 and 100 µg ml−1). Additionally, the leucocyte proliferation assay confirmed polyclonal activation of leucocytes by AREE, indicating the absence of any negative impact of the extract on leucocyte viability. Immune-related genes viz. IL-1β, IFN-γ and C3 showed significantly higher upregulation following incubation of AREE with the leucocytes, indicating enhancement of immune responses at the molecular level. The results collectively suggest the potential of AREE as an effective immunostimulant for use in aquaculture.

Functional characterization of TNF-α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila.

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

Characterization of TCR+ and CD8+ head kidney leucocytes in Japanese flounder (Paralichthys olivaceus) with antisera against TCRα and CD8α.

T lymphocytes play an important role in cellular and adaptive immunity in vertebrates. The mechanisms of the fish immune system are little studied because of the lack of population-specific antibodies. This study examined the expression of two T lymphocyte markers, TCRα (PoTCRα) and CD8α (PoCD8α) in the Japanese flounder (Paralichthys olivaceus). The expression of PoTCRα and PoCD8α was mainly detected in immune/mucosal tissues. Recombinant PoTCRα and PoCD8α were expressed in pET32a and pET259, respectively. Then, rabbit anti-PoTCRα serum and rat anti-PoCD8α serum were prepared. Using serum, the characteristics of TCR+ and CD8+ head kidney leucocytes (HKLs) were investigated. The results of laser scanning confocal microscopy (LSCM) demonstrated that TCRα and CD8α were transmembrane proteins localized on the cell surface. The populations of CD8α- , CD8α+ , TCRα- , and TCRα+ were sorted by flow cytometry (FCM) and analysed using qRT-PCR. The results demonstrated that all TCRα+ /TCRα- or CD8α+ /CD8α- HKLs expressed IFN-γ. The CD4-1 and IgM transcripts were detected only in TCRα- and CD8α- cells. Furthermore, HKL mitogenesis was induced with concanavalin A (ConA) stimulation. Taken together, the results from LSCM and FCM analyses showed that mammalian and P. olivaceus TCR+ and CD8+ leucocytes share basic characteristics.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l−1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l−1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l−1 and 10 mg l−1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l−1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l−1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l−1 though values were significantly similar with concentration from 16 to 128 mg l−1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l−1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Identification and expression analysis of group II C-type lectin domain containing receptors in grass carp Ctenopharyngodon idella.

Group II C-type lectin domain (CTLD) containing receptors belong to a large family of pattern recognition receptors which mainly act on the innate immunity. They are structurally related and consist of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single extracellular CTLD. Although they have been described in teleost fish, their involvement in immune responses is not well understood. In this study, four immune-related lectin-like receptors (termed CiILLR1 and CiILLR5-7), belonging to the group II CTLD receptors, were identified in grass carp (Ctenopharyngodon idella). They contain a short cytoplasmic tail and a single CTLD in the extracellular region. The CiILLR1 has a WxHxxxxxY motif similar to the WxHxxxxY motif which is required for the recognition of β-glucans by some of the group II CTLD containing lectins in mammals. Further, a modified QPD motif (EPD) known to be involved in binding to carbohydrate ligands is present in the CiILLR1, 5 and 6. However, CiILLR7 lacks these motifs. Expression analysis revealed that they were constitutively expressed in the head kidney and spleen. Moreover, CiILLR1, 5 and 6 could be up-regulated in the head kidney and spleen of fish after infection with Flavobacterium columnare and in the primary head kidney leukocytes by LPS and PHA. Expression of CiILLR1, CiILLR5 and CiILLR6 were mainly detected in the enriched lymphocytes whilst CiILLR7 was expressed in the enriched monocytes/macrophages. The results expand existing knowledge on the immune responses of the C-type lectin receptors in teleost fish.

The screen herbal immunopotentiator and research on its effect on the innate immune system and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae

To screen potential herbal immunopotentiators, the effects of 20 herbal aqueous extracts (HAEs) on the oxygen respiratory burst activity (ORBA) and phagocytic activity of tilapia blood leucocytes were determined using nitroblue tetrazolium (NBT) reduction and phagocytizing latex beads methods, respectively. The screened-out herbal aqueous extract (HAE) supplemented diet (10 g/kg, dry herb/dry feed) was consecutively fed to healthy tilapia for in vivo evaluating its effects on the ORBA and phagocytic activity of head-kidney leucocytes, the acid phosphatase (ACP), lysozyme (LZM), catalase (CAT) and superoxide dismutase (SOD) activities of serum, and the mRNA expression levels of IL-β, IL-8, Hsp70, and TNF-α in the spleen. For evaluating the efficacy of the screened-out HAE on preventing streptococcosis, the tilapia were respectively and consecutively fed the control diet and the screened-out HAE supplemented diet for seven and fourteen days and subsequently infected with Streptococcus agalactiae. Their survival cures were compared using the log-rank test. Additionally, the components in the screened-out HAE were identified using ultra-performance liquid chromatography-Q exactive orbitrap-mass spectrometry (UPLC-QE-MS). Results showed that, among the candidate HAEs, Scutellaria baicalensis HAE showed more ability in terms of simultaneously enhancing the ORBA and phagocytic activity of tilapia blood leucocytes as compared to the other candidate HAEs. Oral administration of S. baicalensis HAE significantly elevated the ORBA and phagocytic activity of head-kidney leucocytes, the ACP, LZM, and SOD activities of sera, the mRNA expression levels of IL-8 and Hsp 70, and the cumulative survival rate of tilapia with S. agalactiae infections. The negative and positive models of UPLC-QE-MS identified 256 and 596 components of S. baicalensis HAE, respectively. Taken together, the results of this study indicate that S. baicalensis HAE could prove useful in the prevention of tilapia streptococcosis and suggest that it could be further developed as immunostimulants.

Fish natural killer enhancing factor-A (NKEF-A) enhance cytotoxicity of nonspecific cytotoxic cells against bacterial infection

Natural killer enhancing factor (NKEF)-A/B is a member of Peroxiredoxin (Prxs) family, which is named for the function of enhancing NK cells activity. NKEF also plays essential roles in multiple physiology/pathology processes including inflammation regulation, cancer development and redox reactions. However, the regulatory effects of fish NKEF on immune cells remain largely unknown. In this study, the full-length cDNA of NKEF-A (Accession No. MK584553, designated as On-NKEF-A) was identified from Nile tilapia (Oreochromis niloticus). On-NKEF-A encoded a 198 amino acid peptide with molecular mass of 22.085 kDa. On-NKEF-A protein contained a typical 2-Cys family domain, two active sites (51aa and 172aa) that were conserved in mammals, birds, amphibians and fish. Phylogenetic analysis showed that On-NKEF-A had the closest relationship with Zebra mbuna (Maylandia zebra) NKEF. The On-NKEF-A transcription was present in all examined tissues or organs. And the relative high expression levels of On-NKEF-A was found in head kidney leucocytes and nonspecific cytotoxic cells (NCC). After Streptococcus agalactiae stimulation, On-NKEF-A was significantly up-regulated in head kidney, spleen, gill and skin. Also, On-NKEF-A was markedly induced post S. agalactiae, LPS and poly I:C stimulation in head kidney-derived NCC. Moreover, On-NKEF-A was mainly distributed in cytoplasm of fathead minnow cells (FHM cells). The further in vitro analysis found that the recombinant protein of On-NKEF-A (rOn-NKEF-A) could induce the expression of various molecular markers of B cells, macrophages and NCC, enhanced the cytotoxicity of NCC via increasing the effectors expression. The present data collectively indicate that On-NKEF-A participates in anti-bacterial immune response via regulating NCC activity, which will provide new ideas to further explore the defense mechanism of fish against bacteria.

Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling-An In Vitro Study.

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes’ immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1β and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65. This study showed the ability of methionine surplus to improve leucocytes’ response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.

Green synthesis of gold nanoparticles using Turnera diffusa Willd enhanced antimicrobial properties and immune response in Longfin yellowtail leukocytes

Plant-mediated nanoparticle synthesis is an eco-friendly method designed to reduce toxicity. This research reports the effects of gold nanoparticle green synthesis on immune modulation for the first time, using aqueous extract from Turnera diffusa. First, the chemical composition showed that T. diffussa is abundant in compounds, such as oplopanone, γ-eudesmol, hydroquinone-β-d-glucoside (arbutin) and inositol. The synthesized gold nanoparticles (AuNPDam) were confirmed by ultraviolet visible (UV-Vis) spectrophotometry; the micro-graphical analysis confirmed the average size that was estimated about 24 nm, which were mostly spherical in shape. The Fourier transform infrared spectroscopy (FTIR) technique helped to confirm the functional groups of the synthesized AuNPDam. Its antioxidant capability was analysed using 2,2-diphenyl-1-picrylhydarzyl (DPPH), superoxide radical scavenging and fluorescence recovery after photobleaching (FRAP) methods. These results indicated a worthy antioxidant activity. The synthesized AuNPDam showed strong antibacterial activity against Vibrio parahaemolyticus and Aeromonas hydrophila. Interestingly, AuNPDam were nontoxic to Longfin yellowtail head kidney leukocytes after 24 h. The phagocytosis activity, production of reactive oxygen species and nitric oxide were also enhanced in leukocytes treated with AuNPDam. Overall, the results suggested that AuNPDam is non-cytotoxic, displays strong bactericidal activity and has therapeutic properties by enhancing the immune system.

Nutrient Digestibility, Growth, Mucosal Barrier Status, and Activity of Leucocytes From Head Kidney of Atlantic Salmon Fed Marine- or Plant-Derived Protein and Lipid Sources.

Nutrient digestibility, growth, and mucosal barrier status of fish skin, gills, and distal intestine were studied in Atlantic salmon fed feeds based on marine or plant-derived ingredients. The barrier status was assessed by considering the expression of four mucin genes, five genes that encode antimicrobial proteins, distal intestine micromorphology, and design-based stereology of the midgut epithelium. In addition, the head kidney leukocytes were examined using flow cytometry; to understand the differences in their counts and function. Five experimental feeds containing the main components i) fishmeal and fish oil (BG1), ii) soybean meal (BG2; to induce enteritis), iii) fishmeal as the main protein source and rapeseed oil as the main lipid source (BG3), iv) a mix of plant protein concentrates as the protein sources and fish oil as the lipid source (BG4), and v) plant and marine ingredients in the ratio 70:30 (BG5) were produced for the study. Atlantic salmon with initial weight 72.7 ± 1.2 g was offered the experimental feeds for 65 days. The results revealed that the weights of all fish groups doubled, except for fish fed BG2. Fish fed the BG2 diet had lower blood cholesterol concentration, developed enteritis, had lower expression of muc2 in the distal intestine, and had a compromised barrier status in the intestine. Expression of both the mucin genes and genes that encode antimicrobial peptides were tissue-specific and some were significantly affected by diet. The fish fed BG1 and BG3 had more head kidney lymphocyte-like cells compared to BG5-fed fish, and the phagocytic activity of macrophage-like cells from the head kidney was the highest in fish fed BG1. The intestinal micromorphology and the mucosal mapping suggest two different ways by which plant-based diets can alter the gut barrier status; by either reducing the mucous cell sizes, volumetric densities and barrier status (as noted for BG2) or increasing volumetric density of mucous cells (as observed for BG4 and BG5). The results of the compromised intestinal barrier in fish fed plant ingredients should be further confirmed through transcriptomic and immunohistochemical studies to refine ingredient composition for sustainable and acceptable healthy diets.

Evaluation of an in vitro assay to screen for the immunotoxic potential of chemicals to fish

A wide variety of environmental contaminants has been shown to disrupt immune functions of fish and may compromise their defense capability against pathogens. Immunotoxic effects, however, are rarely considered in ecotoxicological testing strategies. The aim of this study was to systematically evaluate the suitability of an in vitro immuno-assay using selected fish immune parameters to screen for chemicals with known immunotoxic potential and to differentiate them from non-immunotoxicants. Non-stimulated and lipopolysaccharide-stimulated head kidney leukocytes of rainbow trout (Oncorhynchus mykiss) were exposed for 3 h or 19 h to chemicals with different modes of action. As immune parameters, phagocytosis activity, oxidative burst activity and cytokine transcription (IL-1β, TNFα, IL-10) were examined, accompanied by in silico modelling. The immunotoxicants dexamethasone, benzo(a)pyrene, ethinylestradiol and bisphenol A significantly altered the immune parameters at non-cytotoxic concentrations whereas diclofenac had only weak effects. However, the two baseline chemicals with no known immunotoxic potential, butanol and ethylene glycol, caused significant effects, too. From our results it appears that the in vitro fish leukocyte assay as performed in the present study has only a limited capacity for discriminating between immunotoxicants and non-immunotoxicants.

Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus)

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

Effect of chrysophanic acid on immune response and immune genes transcriptomic profile in Catla catla against Aeromonas hydrophila

The effect of chrysophanic acid (CA) (2, 4, and 8 mg kg−1) on the immunity and immune-related gene profile of Catla catla against Aeromonas hydrophila is reported. In both control and treated groups fed with 2 mg kg−1 (2 CA), the phagocytosis, hemolytic, myeloperoxidase content, and superoxide anion production decreased significantly between 6th and 8th weeks, whereas when fed with 4 mg kg−1 CA (4 CA) the H2O2 production and nitric oxide synthase increased significantly between 4th and 8th week. When fed with 2 CA and 4 CA diets, the total protein, bactericidal, and antibody titer increased significantly from the 4th week onwards. When fed with 2 CA, the IL-1β and IL-10 mRNA expression of head kidney leucocytes were significant between weeks 6 and 8. The expressions of toll-like receptors significantly increased when fed with a 4 CA diet from 4th week onwards. The 4 CA group significantly increased in TNF-α, TNF receptor-associated factor 6 (NOD), which influences protein expression, after the 4th week. The mRNA transcription of MHCI, lysozyme-chicken and goose type expressions significantly increased in 4 CA group within the 4th week. In summary, the dietary administration of 4 mg kg−1 of CA (4 CA) provides better immunity and enhances the up-regulation of immune-related genes in Catla against A. hydrophila.

Phenol-rich fulvic acid as a water additive enhances growth, reduces stress, and stimulates the immune system of fish in aquaculture

Aquaculture has become imperative to cover the demands for dietary animal protein. Simultaneously, it has to overcome prejudices from excessive use of antibiotics and environmental impacts. Natural supplements are traditionally applied orally. In this study, we demonstrated another pathway: the gills. Humic substances are immunostimulants and a natural part of every aquatic ecosystem, making them ideal to be used as bath stimulants. Five and 50 mg C/L of a fulvic acid-rich humic substance was added for 28 days to the water of juvenile rainbow trout (Oncorhynchus mykiss). This fulvic acid is characterized by a high content of phenolic moieties with persistent free radicals and a high electron exchange capacity. The high concentration of the fulvic acid significantly increased growth and reduced the food conversion ratio and the response to a handling-stressor. Phagocytosis and potential killing activity of head kidney leukocytes were increased, as well as the total oxyradical scavenging capacity (TOSC) and lysozyme activity in the gills. In conclusion, immunostimulation via gills is possible with our fulvic acid, and the high phenolic content improved overall health and stress resistance of fish.