Establishment and application of four long-term culture cell lines of the olive flounder Paralichthys olivaceus blastocysts.

Four new embryonic cell lines derived from blastocysts of the olive flounder Paralichthys olivaceus, an important commercial marine fish, were established and characterized. They were designated as PoEFCI, PoEFCII, PoEFCIII, and PoEFCIV and were all fibroblastic cells. The cells were cultured in DMEM/F-12 medium supplemented with antibiotics, FBS, and growth factors at temperature of 25 °C and subcultured for >100 passages over 18 months. The origin of the cell lines was confirmed by examining the partial sequences of the cytochrome oxidase c subunit I (COI) gene of the flounder mitochondrial DNA (mtDNA). The four cell lines showed different growth curve patterns. According to the results of gene and protein expression and enzyme activity, the cell lines PoEFCI, PoEFCII, and PoEFC III could be pluripotent. The cells of all four cell lines were also successfully transfected with the green fluorescent protein (GFP) reporter gene, suggesting that they could be used to study gene function in the flounder or other fish. More importantly, PoEFCI-III were sensitive to chromium (Cr) and red sea bream Pagrus major iridovirus (RSIV), so they could be used as a powerful tool for the study of the toxicological investigation of heavy metals and RSIV in fish. Therefore, these cell lines would be useful for biotechnological and toxicological research on marine fish as an in vitro biological system.

Dietary cinnamaldehyde improves muscle protein content by promoting muscle fiber growth via PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling pathway in grass carp (Ctenopharyngodon idella).

Flesh quality is evaluated according to nutritional value and sensory quality. Cinnamaldehyde (CIN) improves mammalian meat quality, but research relating this to aquaculture is scarce. In this study, five doses of CIN (0, 36, 72, 108, 144 mg/kg diet) were fed to grass carp (Ctenopharyngodon idella) for 60 days. The results show that CIN supplementation increased nutritional value by increasing crude protein content. CIN also improved the sensory quality by increasing the pH and collagen content, decreasing shear force, lactate, and cooking loss. These changes may be related to changes in muscle fiber growth by increasing myofiber diameter. The increased myofiber diameter induced by CIN is associated with TOR mRNA and protein levels, and down-regulated FOXO3a mRNA levels, which might be associated with PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling. Based on muscle crude protein content, optimal CIN supplementation dosage was 88.01 mg/kg.

Efficacy of ulvan on immune response and immuno-antioxidant gene modulation in Labeo rohita against columnaris disease.

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg-1 ulvan; the FCR was better (P > 0.05) when fed with 50 mg kg-1 enriched diet. In normal fish fed with or without ulvan supplementation was noted 100% survival rate (SR). In both groups, the red blood cell (RBC) and while blood cell (WBC) counts increased significantly (P > 0.05) when fed with 50 mg kg-1 ulvan diet whereas the hemoglobin (Hb) level increased significantly on being fed with 25 and 50 mg kg-1 ulvan diets. The SOD activity was enhanced significantly in both groups fed with any dose of ulvan diets whereas the MDA and GPx activity increased only with 25 and 50 mg kg-1 ulvan diets. The phagocytic (PC) activity significantly increased with any enriched diet and control diet groups while the respiratory burst (RB) activity increased only with 50 mg kg-1 ulvan diet. The alternate complement pathway (ACP), activity of lysozyme (Lyz), and immunoglobuline M (IgM) were better in both groups fed with 50 mg kg-1 ulvan diet. The SOD and GPx antioxidant gene expression were significantly high in both groups fed with any ulvan diet while the Nrf2 gene expression was high with 50 mg kg-1 ulvan diet. The IL-1ß, TNFα, hepcidin, Lyz, and IgM cytokines or proteins mRNA expression were significant in both groups fed with all ulvan supplement diet whereas the ß-2M expression was significant only with 50 mg kg-1 ulvan diet. The present research indicates that both L. rohita groups fed with 50 mg kg-1 ulvan diet significantly improved growth, antioxidant system, immune defense system, and immuno-antioxidant related gene expression against F. columnaris.

Responses to Mineral Supplementation and Salmon Lice (Lepeophtheirus salmonis) Infestation in Skin Layers of Atlantic Salmon (Salmo salar L.).

The crustacean ectoparasite salmon louse (Lepeophtheirus salmonis), which severely affects Atlantic salmon health and welfare is one of the main problems of commercial aquaculture. In the present study, fish were fed a diet supplemented with extra minerals through the inclusion of a commercial additive (Biofeed Forte Salmon), substituting wheat in the control diet, before experimental infestation with salmon lice. Lice counts reduced with time but with no apparent effect of the diets. Further, fish fed the mineral diet had an overall higher number of blue (acidic) mucous cells, while the ratio of purple mucous cells was higher in the mineral diet. The transcriptional response in skin was enhanced at 7 dpc (copepodite life stage) in fish fed the mineral diet including immune and stress responses, while at 21 dpc (pre-adult life stage), the difference disappeared, or reversed with stronger induction in the control diet. Overall, 9.3% of the genes affected with lice also responded to the feed, with marked differences in outer (scale + epidermis) and inner (dermis) skin layers. A comparison of transcriptome data with five datasets from previous trials revealed common features and gene markers of responses to lice, stress, and mechanically induced wounds. Results suggested a prevalence of generic responses in wounded skin and lice-infected salmon.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Molecular characterization and antiparasitic activity analysis of a novel piscidin 5-like type 4 from Larimichthys crocea.

Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture teleosts and cause high lethality and economic loss, especially Larimichthys crocea. Current methods of controlling or preventing this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. The antiparasitic activity of some antimicrobial peptides (AMPs) attracted extensive attention of scholars. In the study, a novel piscidin 5-like type 4 (termed Lc-P5L4) excavated from comparative transcriptome of C. irritans - immuned L. crocea was identified and characterized. Sequence analysis shows the full-length cDNA of Lc-P5L4 is 539 bp containing an open reading frame (ORF) of 198 bp which encodes a peptide of 65 amino acid residues. The genome consists of three exons and two introns which exist in its ORF, and all the exon-intron boundaries are in accordance with classical GT-AG rule (GT/intron/AG). Multiple alignments indicate the signal peptides share highly conserved identity, while mature peptides are more diverse. Phylogenetic analysis displays Lc-P5L4 clusters together with other members of piscidin 5-like family. Next, quantitative Real-time PCR (qRT-PCR) detection found C. irritans infection could upregulate Lc-P5L4 expression level in all tested tissues significantly, it appeared earliest upregulation in the theronts infection stage in the head kidney; the expression contents reached to maximum level in the intestine, gill and muscle during trophonts falling off stage; while it was just upregulated during secondary bacterial infection stage in the liver and spleen. The data showed Lc-P5L4 upregulation time points were in accordance with different infection stages. With recombinant Lc-P5L4 (rLc-P5L4) obtained through Escherichia coli system, in vitro assay showed rLc-P5L4 could cause cilia deactivation, cell bodiesclumping and sticking to each other, then cell membrane rupture and contents leakage. The data illustrated Lc-P5L4 played critical roles in the immune defense against C. irritans infection, and provided another proof that piscidins exhibit multiple anti- C. irritans features.

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii.

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation-reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.

Bacillus subtilis H2 modulates immune response, fat metabolism and bacterial flora in the gut of grass carp (Ctenopharyngodon idellus).

Functional ingredients such as Bacillus subtilis are used in aquaculture to improve fish condition, modulate microbiota and promote a healthy intestinal system. However, the underlying mechanisms of grass carp treated with B. subtilis are not fully characterized. This study investigated the gut microbes of grass carp after treated with B. subtilis H2 (106 CFU/mL) and Aeromonas hydrophila (106 CFU/mL). The intestinal flora was found that the dominant bacterial phyla identified in all samples were Proteobacteria, Actinobacteria, Fusobacteria, Bacteroidetes and Acidobacteria. Compared with the control group, the relative abundance of Proteobacteria and Bacteroidetes in B. subtilis group were significantly increased. In addition, the relative abundances of Aeromonas and Shewanella in A. hydrophila group were more than the control group. For the intestinal transcriptomic profiling of the grass carp treated with B. subtilis H2, 824 different expressed genes (DEGs) between the B. subtilis H2 treated and non-treated groups were detected, including 365 up-regulated and 459 down-regulated genes. Six DEGs were randomly selected for further validation by quantitative real-time RT-PCR (qRT-PCR) and the results were consistent with the RNA-seq data. Additionally, eight immunomodulatory genes (IL-4, IL-11, IFN-α, CSF, FOSB, MAPK12b, IGHV3-11 and IGHV3-21) were significantly up-regulated after treated with B. subtilis H2. Furthermore, almost all the lipid metabolism-associated genes were significantly up-regulated after treated with B. subtilis H2 according to the lipid metabolism pathways. Eleven lipid metabolism-associated genes were selected by qRT-PCR, which showed that the expressions of almost all the selected genes were increased, especially Apob-48, ABCG8 and DGAT. Taken together, our results support that B. subtilis could modulate the immune response, fat metabolism and bacterial assembly in the gut of grass carp.

Dietary Scenedesmus ovalternus improves disease resistance of overwintering gibel carp (Carassius gibelio) by alleviating toll-like receptor signaling activation.

This study was conducted to investigate the effect of dietary Scenedesmus ovalternus on the growth and disease resistance of gibel carp (Carassius gibelio) during overwintering. Gibel carp (initial body weight: 90.39 ± 0.33 g) were fed with diets containing 0% or 4% Scenedesmus ovalternus (DS0 and DS4) for 4 weeks during the early overwintering period, and then all fish were left unfed during the late overwintering period. A bacterial challenge test using Aeromonas hydrophila was subsequently conducted. The 4% Scenedesmus ovalternus diet had no effect on the growth of gibel carp (P > 0.05), but did improve the survival rate after the challenge (P ≤ 0.05). In the DS0 group, the bacterial challenge decreased the contents of complement 3 (C3), immunoglobulin M (IgM), interleukin 2 (IL2) and tumor necrosis factor α (TNFα) in fish (P < 0.05); in the DS4 group, the challenge increased total antioxidant capacity (T-AOC) and myeloperoxidase (MPO) activity but decreased IL2 and TNFα contents (P < 0.05). The activities of MPO and contents of C3, IgM and TNFα were higher in the DS4 group than that fed the DS0 diet after bacterial challenge (P < 0.05). Compared to pre challenge, the expression levels of toll like receptor 2 (TLR2), toll like receptor 3 (TLR3), toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), TIR-domain-containing adapter-inducing interferon ß (TRIF), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα), transforming growth factor ß (TGFß), interleukin 1ß (IL1ß), tumor necrosis factor α1 (TNFα1) and interleukin 10 (IL10) in the head kidney of gibel carp were induced after challenge (P < 0.05). Gibel carp fed the DS4 diet showed lower expression of TGFß in head kidney before the challenge and lower expression of TLR2, TLR3, TLR4, TIRAP, TRIF, IκBα, TNFα1, IL10 and TGFß after the challenge than that fed the DS0 diet (P < 0.05). Overall, Scenedesmus ovalternus supplement enhanced the resistances of gibel carp against A. hydrophila after overwintering via the TLR signaling pathway.

Effects of dietary yeast hydrolysate on the growth, antioxidant response, immune response and disease resistance of largemouth bass (Micropterus salmoides).

A 56-day growth trial was conducted to investigate the effects of dietary yeast hydrolysate on the growth performance, antioxidation, immune response and resistance against Aeromonas hydrophila in largemouth bass. Four experimental diets were prepared with yeast hydrolysate levels of 0% (Y0), 1.5% (Y1.5), 3.0% (Y3.0) and 4.5% (Y4.5). Each diet was randomly assigned to triplicate 150-L tanks and each tank was stocked with 30 largemouth bass (initial body weight, IBW = 7.71 ±â€¯0.02 g). A challenge test was carried out after the feeding trial by injecting A. hydrophila intraperitoneally for 4-day observation. The results showed that the FBW and WGR in Y1.5 group were significantly higher than those in Y0 group (P < 0.05) and the feed conversion ratio (FCR) got the lowest value in Y1.5 group. And the hydrolysate supplement significantly increased the 4-day cumulative survival rate after the bacterial challenge (P < 0.05). The plasma malondialdehyde was lower in the yeast hydrolysate supplement groups in both pre- and post-challenge test (P < 0.05), while the plasma C3 increased (P < 0.05). In post-challenge test, the plasma superoxide dismutase (SOD) and catalase (CAT) activities increased in the Y1.5 and Y3.0 groups respectively (P < 0.05), and plasma lysozyme in Y1.5 group and the plasma IgM in Y3.0 group were higher than those in others respectively (P < 0.05). For the q-PCR results, in post-challenge test, the hepatic hep2 expression level in Y1.5 and Y4.5 groups were both significantly higher than those in others (P < 0.05), as well as il-8 in Y3.0 group. The spleen hif-1alpha and tgf-beta1 expression levels in Y4.5 group were all significantly lower than those in others (P < 0.05), while the gilt was significantly higher (P < 0.05) in the post-challenge test. And the expression levels of spleen tnf-alpah1 in Y1.5 and Y3.0 groups and il-8 in Y3.0 group were all significantly higher than those in other groups (P < 0.05) in the post-challenge test. The head kidney gilt expression level was significantly higher in the yeast hydrolysate supplement groups compared with the Y0 group (P < 0.05), and the head kidney il-8 expression level in Y1.5 group was significant higher than those in other groups in post-challenge test (P < 0.05). The present results indicated dietary yeast hydrolysate improved the antioxidant ability and enhanced the immune response of largemouth bass without negative effect on growth. And 1.5% or 3.0% of dietary yeast hydrolysate was recommended for largemouth bass based on the present results.

Impact and consequences of dietary riboflavin deficiency treatment on flesh quality loss in on-growing grass carp (Ctenopharyngodon idella).

Fish is among the cheapest and most promising sources of animal protein. The main edible portion of fish is muscle. This study explored the impact of dietary riboflavin on fish flesh quality and showed the possible role of muscle antioxidant defense in flesh quality in relation to dietary riboflavin. On-growing grass carp (initial average weight of 275.82 ± 0.57 g) were fed diets containing graded levels of riboflavin (0.63, 1.95, 3.98, 6.02, 7.96, and 10.04 mg kg-1 diet) for eight weeks. The results indicated that compared with the optimal riboflavin levels (3.98 and/or 6.02 mg riboflavin per kg diet), riboflavin deficiency treatment (0.63 mg riboflavin per kg diet) significantly reduced the muscle nutrients, including the protein, lipid, flavor amino acid, and total essential amino acid contents. Furthermore, the muscle shear force, pH value, and hydroxyproline concentration were reduced, while the muscle cooking loss and lactic acid content increased (P < 0.05). Compared with optimal riboflavin levels, the riboflavin deficiency treatment increased the reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl contents, while riboflavin treatments of 3.98-10.04 mg riboflavin per kg diet showed the lowest ROS and MDA contents (P < 0.05). Compared with the optimal riboflavin levels, the riboflavin deficiency treatment decreased the activities of copper/zinc superoxide dismutase (CuZnSOD), glutathione reductase (GR), catalase (CAT), and glutathione peroxidase (GPx), and reduced the glutathione (GSH) content (P < 0.05). Furthermore, the relative mRNA levels of antioxidant enzymes, including CuZnSOD, CAT, GR and GPx, and antioxidant-related signaling molecules, including NF-E2-related factor 2 (Nrf2) and casein kinase 2, were down-regulated, while those of Kelch-like ECH-associated protein 1b were up-regulated (P < 0.05). Collectively, the present study indicates that riboflavin deficiency treatment reduces the flesh quality, partly due to inhibition of the antioxidant defense through the Nrf2 signaling pathway, while optimal riboflavin levels reverse these negative effects.

Effects of Vitamin D2 (Ergocalciferol) and D3 (Cholecalciferol) on Atlantic Salmon (Salmo salar) Primary Macrophage Immune Response to Aeromonas salmonicida subsp. salmonicida Infection.

Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D2 and D3 through the diet. In mammals, vitamin D forms are involved in mineral metabolism, cell growth, tissue differentiation, and antibacterial immune response. Vitamin D is an essential nutrient in aquafeeds for finfish. However, the influence of vitamin D on fish cell immunity has not yet been explored. Here, we examined the effects of vitamin D2 and vitamin D3 on Salmo salar primary macrophage immune response to A. salmonicida subspecies salmonicida infection under in vitro conditions. We determined that high concentrations of vitamin D2 (100,000 ng/ml) and D3 (10,000 ng/ml) affect the growth of A. salmonicida and decrease the viability of S. salar primary macrophages. In addition, we determined that primary macrophages pre-treated with a biologically relevant concentration of vitamin D3 for 24 h showed a decrease of A. salmonicida infection. In contrast, vitamin D2 did not influence the antibacterial activity of the S. salar macrophages infected with A. salmonicida. Vitamin D2 and D3 did not influence the expression of canonical genes related to innate immune response. On the other hand, we found that A. salmonicida up-regulated the expression of several canonical genes and suppressed the expression of leukocyte-derived chemotaxin 2 (lect-2) gene, involved in neutrophil recruitment. Primary macrophages pre-treated for 24 h with vitamin D3 counteracted this immune suppression and up-regulated the transcription of lect-2. Our results suggest that vitamin D3 affects A. salmonicida attachment to the S. salar primary macrophages, and as a consequence, the A. salmonicida invasion decreased. Moreover, our study shows that the positive effects of vitamin D3 on fish cell immunity seem to be related to the lect-2 innate immunity mechanisms. We did not identify positive effects of vitamin D2 on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D3, cholecalciferol, induced anti-bacterial innate immunity pathways in Atlantic salmon primary macrophages, suggesting that its utilization as a component of a healthy aquafeed diet in Atlantic salmon could enhance the immune response against A. salmonicida.

Omega-3 polyunsaturated fatty acids alleviate hepatic steatosis-induced inflammation through Sirt1-mediated nuclear translocation of NF-κB p65 subunit in hepatocytes of large yellow croaker (Larmichthys crocea).

Hepatic steatosis induced inflammation is becoming increasingly prevalent in farmed fish. This study was conducted to investigate the protective effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) against hepatic steatosis-induced inflammation and its potential molecular mechanisms in hepatocyte of large yellow croaker (Larmichthys crocea). We found that the hepatic steatosis-induced inflammation was relieved by ω-3 PUFAs, meanwhile, the Sirt1 activity and transcript expression was increased by ω-3 PUFAs. The increased Sirt1 activity can decrease the hepatic steatosis-induced inflammation. The protective effects of ω-3 PUFAs against hepatic steatosis-induced inflammation was reversed by the treatment with Sirt1 inhibitor EX-527. The nuclear translocation of nuclear transcription factor kappa-B (NF-κB) p65 was significantly decreased after ω-3 PUFAs treatments compared to the palmitic acid stimulation group. The ω-3 PUFAs induced cytoplasm translocation of NF-κB p65 was reversed by EX-527. Together, ω-3 PUFAs alleviate hepatic steatosis-induced inflammation through Sirt1-mediated nuclear translocation of NF-κB p65 subunit in hepatocytes of large yellow croaker. The present study provides important insight into the mechanisms of the protective effects of ω-3 PUFAs, providing theory bases for alleviating the hepatic steatosis induced inflammation of farmed fish, thereby offering great benefits to the aquaculture industry and fish consumers.

Molecular Identification of Multiple Antibiotic Resistant Fish Pathogenic Enterococcus faecalis and their Control by Medicinal Herbs.

The opportunistic fish pathogen, Enterococcus faecalis has been reported to cause mass mortality in several fish species in different countries. The objectives of this study were to (i) identify E. faecalis from the diseased fishes through molecular techniques; (ii) assess the antibiotic susceptibility profile of E. faecalis isolates; and (iii) control disease in tilapia fish by treatment with medicinal plant extracts. A total of 48 isolates were phenotypically identified as Enterococcus species from tilapia, stinging catfish and walking catfish cultivated in several fish farms in Gazipur. Ten randomly selected isolates were identified as E. faecalis by 16S rRNA gene sequencing. Artificial infection revealed that most of the isolates caused moderate to high mortality in fishes with characteristic disease symptoms. These isolates exhibited resistance to multiple antibiotics in vitro. Bioassay revealed that organic extracts of Tamarindus indica and Emblica officinalis leaves, Allium sativum bulb, and Syzygium aromaticum bud inhibited the growth of E. faecalis. Methanol extracts of A. sativum and methanol and acetone extracts of S. aromaticum significantly reduced the mortality of fish artificially infected with E. faecalis as both preventive and therapeutic agents. This is the first report on molecular identification, and herbal control of fish pathogenic E. faecalis in Bangladesh.

Dietary zinc deficiency reduced growth performance, intestinal immune and physical barrier functions related to NF-κB, TOR, Nrf2, JNK and MLCK signaling pathway of young grass carp (Ctenopharyngodon idella).

Our study investigated the effects of dietary zinc (Zn) deficiency on growth performance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (244.14 ± 0.40 g) were fed graded levels of zinc lactate (10.71, 30.21, 49.84, 72.31, 92.56, 110.78 mg Zn/kg diet) and one zinc sulfate group (56.9 mg Zn/kg diet) for 60 days. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. These results indicated that compared with optimal dietary Zn level, dietary Zn deficiency (10.71 mg/kg diet) decreased the production of antibacterial compounds, up-regulated pro-inflammatory cytokines related to nuclear factor kappa B (NF-κB) and down-regulated anti-inflammatory cytokines related to target of rapamycin (TOR) in three intestinal segments of young grass carp (P < 0.05), suggesting that dietary Zn deficiency could impair intestinal immune barrier of fish; decreased the activities and mRNA levels of antioxidant enzymes related to NF-E2-related factor 2 (Nrf2), up-regulated the mRNA levels of caspase-3, -7, -8, -9 related to p38 mitogen activated protein (p38 MAPK) and c-Jun N-terminal protein kinase (JNK), down-regulated the mRNA levels of tight junction complexes (TJs) related to myosin light chain kinase (MLCK) in three intestinal segments of young grass carp (P < 0.05), demonstrating that dietary Zn deficiency could injury intestinal physical barrier of fish. Besides, the Zn requirements (zinc lactate as Zn source) based on percent weight gain (PWG), against enteritis morbidity, acid phosphatase (ACP) activity in the proximal intestine (PI) and malondialdehyde (MDA) content in the PI of young grass carp was estimated to be 61.2, 61.4, 69.2 and 69.5 mg/kg diet, respectively. Finally, based on specific growth rate (SGR), feed efficiency (FE) and against enteritis morbidity of young grass carp, the efficacy of zinc lactate relative to zinc sulfate were 132.59%, 135.27% and 154.04%, respectively.

Comparative analysis of two ferritin subunits from blunt snout bream (Megalobrama amblycephala): Characterization, expression, iron depriving and bacteriostatic activity.

Iron is an essential microelement for almost all living organisms, while an excess of iron is toxic, thus maintenance of iron homeostasis is vital. As iron storage protein, ferritin plays an important role in iron metabolism. In the present study, we cloned and characterized the ferritin H subunit from Megalobrama amblycephala, termed as MamFerH. An iron-responsive element (IRE) was predicted in the 5' untranslated region (UTR) of MamFerH, while its bulge structural was different from that of the reported ferritin M subunit (MamFerM). The MamFerH and MamFerM genes exhibited similar expression patterns during early development with specifically high expression post hatching, whereas their tissue expression patterns were different. Specifically, MamFerM was highly expressed in the spleen, liver and kidney, while MamFerH was predominantly expressed in the blood and brain, indicating their different functions. In addition, the expression of the two genes was induced upon Aeromonas hydrophila infection at both transcriptional and translational levels, and MamFerH was more efficient. Immunohistochemistry and immunofluorescence analysis confirmed their significant changes at protein level and distribution in the liver post infection, indicating their participation in host immune response. Furthermore, bacteriostatic experiment revealed that recombinant MamFerH displayed more significant inhibitory effect on the growth of A. hydrophila.

Plasma immune protein analysis in the orange-spotted grouper Epinephelus coioides: Evidence for altered expressions of immune factors associated with a choline-supplemented diet.

This study aimed to unravel the regulatory roles of choline in activating immune responses and disease resistance of the orange-spotted grouper Epinephelus coioides. Fish were fed a choline-supplemented diet at 1 g kg-1 of feed for 30 days. Fish fed a fish meal basal diet without choline-supplement served as controls. At the end of the feeding trial, fish were challenged with Vibrio alginolyticus. Meanwhile, plasma proteomics of fish in each group were also evaluated by two-dimensional gel electrophoresis (2-DE), and differentially expressed proteins were identified by tandem mass spectrophotometry (MS/MS), then a Western blot analysis or real-time polymerase chain reaction was used to confirm differential expressions of immune-enhancing proteins. Results showed that choline significantly increased survival of E. coioides 48 days after being injected with V. alginolyticus. From maps of plasma proteins, a comparative analysis between the control and choline groups revealed that 111 spots matched, with 26 altered expression spots in the choline group. Of these 26 spots, 16 were upregulated and 10 downregulated. After protein identification by reverse-phase nano-high-performance liquid chromatography-electrospray ionization MS/MS analysis, eight of 26 proteins were found to be immune-related proteins, all of which were upregulated, including complement 3 (C3), alpha-2-macroglobulin-P-like isoform (A2M), fibrinogen beta chain precursor (FBG), and immunoglobulin heavy constant mu (Ighm) proteins. Expression of the A2M protein and A2M enzyme activity in plasma of fish fed choline significantly increased compared to the control group. Additionally, A2M messenger (m)RNA transcripts were also upregulated in the liver and kidneys. Significantly higher C3 expressions at both the mRNA and protein levels were detected in the liver of fish in the choline group. Moreover, FBG gene expressions in the liver and kidneys significantly increased, while Ighm increased in the kidneys and spleen of fish in the choline group. Our results suggest that dietary administration of choline can protect grouper against bacterial infections through activating the complement system, thereby inducing antiprotease activity and natural antibodies that play important roles in the innate immune system of fish.

Methionine hydroxy analogue improves intestinal immunological and physical barrier function in young grass carp (Ctenopharyngodon idella).

This study was conducted to test the hypothesis that methionine hydroxy analogue (MHA) enhances the defense against enteritis occurrence via improving intestinal barrier function in fish. After 630 young grass carp (Ctenopharyngodon idella) (259.70 ± 0.47 g) fed six graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one dl-methionine group (6.4 g/kg diet) for 8 weeks. At the end of feeding trial, 15 fish from each treatment were challenged with Aeromonas hydrophila for 14 days. The results indicated that optimal MHA enhanced the capacity of fish against enteritis emergence, which might be related to the positive effects of MHA on intestinal immunological and physical barrier function in fish. Dietary MHA supplementation enhanced intestinal immunological barrier function via (1) lysozyme (LZM) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin (head kidney), ß-defensin-1; (2) repressing p38MAPK/IKKß/IκBα/NF-κB signaling pathway to down-regulate pro-inflammatory cytokines mRNA levels except IL-8 mRNA level only in mid and distal intestine; (3) potentiating TOR-signal cascades to up-regulate anti-inflammatory cytokines. Meanwhile, dietary MHA supplementation improved intestinal physical barrier via (1) down-regulating c-Jun N-terminal kinase mRNA levels to inhibit death receptor and mitochondria pathways induced apoptosis; (2) modulating Keap1a/Nrf2 system to elevate antioxidant enzymes genes isoforms mRNA levels and corresponding enzymes activities, subsequently alleviate oxidative damage; (3) down-regulating MCLK gene expression to up-regulating occludin, zonula occluden 1 and claudins mRNA levels except claudin-7a and claudin-7b only in the proximal intestine. In conclusion, bases on the capacity defense against enteritis, proximal intestinal malondialdehyde content and lysozyme activity, the optimal MHA supplementation levels were 5.83, 5.59 and 6.07 g/kg diet (4.01 g/kg methionine basal), respectively. This study indicates that MHA exerts a positive effect on fish intestinal health status and a superior efficacy to dl-methionine based on the positive effects.

Antiviral effects of extracts from Celosia cristata and Raphanus sativus roots against viral hemorrhagic septicemia virus.

The antiviral activity of an extract mixture from Celosia cristata and Raphanus sativus was tested against viral hemorrhagic septicemia virus (VHSV). Pretreatment of EPC cells with this extract up to 72 h before VHSV infection markedly reduced the virus titer, but it had no effect when added after virus inoculation. In olive flounder that received 5 µg of extract per fish, Mx expression peaked at 48 h after treatment. In contrast, ISG15 and TLR2 expression peaked at 72 h, and that of TLR7 peaked at 48 h, followed by a slight decrease at 72 h, indicating that the antiviral activity was mediated by induction of gene expression involved in the innate immune response.

Dietary ß-glucans differentially modulate immune and stress-related gene expression in lymphoid organs from healthy and Aeromonas hydrophila-infected rainbow trout (Oncorhynchus mykiss).

Although ß-glucans stimulating effects have already been demonstrated on the immune system of numerous animal species, available data remain relatively variable and more research should be done regarding the complexity of underlying mechanisms. In this context, the present study aimed to evaluate the stress and immune-related effects of dietary ß-glucans (i.e. Macrogard®) by considering a number of influencing factors such as the dose (0, 0.1, 0.2 and 0.5% in food), feeding duration (15 versus 30 days), tissue (blood, kidney, spleen, gills) and infection status (healthy or infected). Blood parameters (lysozyme, ACH50 activities, leucocyte populations) and mRNA expression level of several immune- and stress-related genes (TFN-α1, IL-1ß, IL10, COX-2, TGF-ß, MC2R, HSP70) were measured. Our results suggest that spleen may be a highly responsive organ to dietary ß-glucans both in healthy or infected fish, and that this organ may therefore significantly contribute to the immune reinforcement induced by such immunostimulatory diet. Our study further reveals that overdoses of ß-glucans and/or prolonged medication can lead to a non-reactive physiological status and, consequently, to a poor immune response. All in all, the current data emphasizes the need for further extensive research in the field of dietary ß-glucans as a preventive method for farmed fish protection.