Probiotics, prebiotics and synbiotics improved the functionality of aquafeed: Upgrading growth, reproduction, immunity and disease resistance in fish.

Aquaculture plays an increasingly significant role in improving the sustainability of global fish production. This sector has been intensified with the advent of new husbandry practices and the development of new technology. However, the increasing intensification and indiscriminate commercialized farming has enhanced the vulnerability of cultivated aquatic species to damage from pathogens. In efforts to confront these various diseases, frequent use of drugs, antibiotics, chemotherapeutics, and agents for sterilization have unintentionally added to the risk of transmission of pathogens and harmful chemical compounds to consumers. Some natural dietary supplements are believed to have the potential to offset this setback in aquaculture. Application of bio-friendly feed additives such as probiotics, prebiotics and synbiotics are becoming popular dietary supplements with the potential to not only improve growth performance, but in some cases can also enhance immune competence and the overall well-being of fish and crustaceans. The present review discusses and summarizes the effects of probiotics, prebiotics and synbiotics application on growth, stress mitigation, microbial composition of intestine, immune system and health condition of aquatic animals in association with existing constraints and future perspectives in aquaculture.

Dietary plant pigment on blood-digestive physiology, antioxidant-immune response, and inflammatory gene transcriptional regulation in spotted snakehead (Channa punctata) infected with Pseudomonas aeruginosa.

The current study addressed to investigate the effect of lycopene (LYC) on blood physiology, digestive-antioxidant enzyme activity, specific-nonspecific immune response, and inflammatory gene transcriptional regulation (cytokines, heat shock proteins, vitellogenins) in spotted snakehead (Channa punctata) against Pseudomonas aeruginosa. In unchallenged and challenged fish treated with 200 mg LYC enriched diet the growth performance and digestive-antioxidant enzymes increased after 30 days, whereas with inclusion of 100 or 400 mg LYC in the diets, the increase manifested on or after 45 days. No mortality in fish treated with any LYC diet against P. aeruginosa was revealed. In the unchallenged and challenged fish the phagocytic (PC) activity in head kidney (HK) and spleen were significantly enhanced when fed the control diet or other LYC diets, whereas the respiratory burst (RB) activity and nitric oxide (NO) production significantly increased when fed the 200 mg diet for 45 and 60 days. Similarly, the lysozyme (Lyz) activity in the HK and spleen, and total Ig content in serum were significantly higher in both groups fed the 200 mg LYC diet for 15, 45, and 60 days. Heat shock protein (Hsp 70) was significantly improved in the uninfected group fed the 200 mg LYC diet for 45 and 60 days, but Hsp27 did not significantly change among the experimental groups at any time points. TNF-α and IL-6 mRNA pro-inflammatory cytokine expression significantly increased in both groups fed the 200 mg LYC diet after 45 and 60 days, while the IL-12 mRNA expression was moderate in both groups fed the same diet for 60 days. The IL-10 did not significant mRNA expression between groups at any sampling. The iNOS and NF-κB mRNA expression was pointedly high in both groups fed the 200 mg LYC diet on day 45 and 60. Vitellogenin A (VgA) mRNA was significantly higher in the uninfected fish fed the 100 and 200 mg LYC diets for 45 and 60 days, but VgB did not reveal significant difference between the treatment groups at any time points. The present results suggest that supplementation of LYC at 200 mg significantly modulate the blood physiology, digestive-antioxidant enzymes, specific-nonspecific immune parameters, and cytokines, Hsp, and vitellogenins in spotted snakehead against P. aeruginosa.

Phytogenic Bioactive Compounds Shape Fish Mucosal Immunity.

Aquaculture growth will unavoidably involve the implementation of innovative and sustainable production strategies, being functional feeds among the most promising ones. A wide spectrum of phytogenics, particularly those containing terpenes and organosulfur compounds, are increasingly studied in aquafeeds, due to their growth promoting, antimicrobial, immunostimulant, antioxidant, anti-inflammatory and sedative properties. This trend relies on the importance of the mucosal barrier in the fish defense. Establishing the phytogenics' mode of action in mucosal tissues is of importance for further use and safe administration. Although the impact of phytogenics upon fish mucosal immunity has been extensively approached, most of the studies fail in addressing the mechanisms underlying their pharmacological effects. Unstandardized testing as an extended practice also questions the reproducibility and safety of such studies, limiting the use of phytogenics at commercial scale. The information presented herein provides insight on the fish mucosal immune responses to phytogenics, suggesting their mode of action, and ultimately encouraging the practice of reliable and reproducible research for novel feed additives for aquafeeds. For proper screening, characterization and optimization of their mode of action, we encourage the evaluation of purified compounds using in vitro systems before moving forward to in vivo trials. The formulation of additives with combinations of compounds previously characterized is recommended to avoid bacterial resistance. To improve the delivery of phytogenics and overcome limitations associated to compounds volatility and susceptibility to degradation, the use of encapsulation is advisable. Besides, newer approaches and dedicated methodologies are needed to elucidate the phytogenics pharmacokinetics and mode of action in depth.

Effects of dietary Astragalus Propinquus Schischkin polysaccharides on growth performance, immunological parameters, antioxidants responses and inflammation-related gene expression in Channa argus.

The present study investigated the effects of dietary Astragalus Propinquus schischkin polysaccharides on growth, immune responses, antioxidants responses and inflammation-related genes expression in Channa argus. Channa argus were randomly divided into 5 groups and fed 5 levels diets of A. propinquus schischkin polysaccharides (0, 250, 500, 1000 and 2000 mg/kg) for 56 days. The results showed, dietary A. propinquus schischkin polysaccharides addition can increase the final body weight, weight gain and specific growth rate, decrease the feed conversion ratio of Channa argus. And dietary A. propinquus schischkin polysaccharides supplementation can increase the levels of serum superoxide dismutase, catalase, glutathione peroxidase, lysozyme, complement 3, complement 4, immunoglobulin M and alkaline phosphatase, decrease the levels of serum malondialdehyde, cortisol, aspartate aminotransferase and glutamic-pyruvic transaminase. Furthermore, dietary A. propinquus schischkin polysaccharides can decrease the gene expression levels of interleukin-1ß, interleukin-, tumor necrosis factor-α and nuclear factor-κB, increase the gene expression levels of glucocorticoid receptor in liver, spleen, kidney, intestine. To sum up, dietary A. propinquus schischkin polysaccharides can accelerate growth, enhance immune responses and antioxidants responses, regulate inflammation-related genes expression in Channa argus and the optimum amount is 1000 mg/kg.

Exploring the garlic (Allium sativum) properties for fish aquaculture.

The aquaculture industry's rapid growth to meet commercial demand can trigger an outbreak of infectious diseases due to high-density farming. Antibiotic overuse and misuse in fish farming and its global health consequences have led to searching for more natural alternatives such as medicinal plants. In this sense, garlic (Allium sativum) has different bioactive compounds with biological properties for animal health. Among them are the ajoene, alliin, and allicin, which confer biological properties such as growth promotion, antimicrobial, antiviral, antioxidant, and antiparasitic. Ways to use garlic in aquaculture include oil, fresh mash, aqueous extract, and garlic powder. The powder presentation is the most used in aquaculture; it is generally applied by oral administration, adding to the feed, and the dose used ranges from 0.05 to 40 g/kg of feed. Garlic has been used in the aquaculture of different species such as rainbow trout (Oncorhynchus mykiss), spotted grouper (Epinephelus coioides), catfish (Clarias gariepinus), tilapia (Oreochromis niloticus), guppy fish (Poecilia reticulata), goldfish (Carassius auratus), and barramundi (Lates calcarifer). In addition to its properties, garlic's usage became popular, thanks to its low cost, easy incorporation into food, and little environmental impact. Therefore, its application can be an effective solution to combat diseases, improve organisms' health using natural supplies, and as an alternative to antibiotics. This review reports and discusses plant-derived products' beneficial properties, emphasizing garlic and its usages in fish aquaculture.

Combined effects of enzymes and probiotics on hemato-biochemical parameters and immunological responses of juvenile Siberian sturgeon (Acipenser baerii).

A 10-week feeding trial was run to investigate the separate and simultaneous effects of exogenous enzymes (Enz), probiotics (Pro), and Pro-Enz mixtures on the hematology indices, serum biochemical parameters, and innate-immunity status of juvenile Siberian sturgeon. The fish (138.06 ± 3.64 g) were randomly dispersed into 12 tanks (20 individuals per tank) and fed with Enz (Phytase, protease, and xylanase), Pro (Pediococcus pentosaceus and Lactococcus lactis), and Pro-Enz cocktail. At the end of the feeding bioassay, the highest values of red blood cell count, hemoglobin concentration, hematocrit level, and lymphocyte percentage followed by the lowest neutrophil percentage were obtained in Pro-Enz treatment (P < 0.05). Despite a significantly lower level of alkaline phosphatase in the fish fed with Pro supplemented diet (P < 0.05), no significant difference was found in the serum level of alanine aminotransferase and aspartate aminotransferase among the experimental groups (P > 0.05). Total protein content was significantly upregulated in serum and skin mucus samples from those fed with supplemented diets compared to the control group (P < 0.05). In both serum and skin mucus samples, higher immune responses in terms of lysozyme activity, immunoglobulin M, total protein was seen in Pro-Enz treatment compared to the control group followed by the serum complement components (P < 0.05). The results indicate that the combinational supplementation of Siberian sturgeon diet with the exogenous enzymes and probiotics modulates the physiometabolic responses and innate immune system to a higher grade than their individual supplementation.

Effects of dietary leucine levels on intestinal antioxidant status and immune response for juvenile golden pompano (Trachinotus ovatus) involved in Nrf2 and NF-κB signaling pathway.

The aim of the study was to evaluate the effects of dietary leucine level on growth performance, intestinal antioxidant status and immune response involved in Nrf2 and NF-κB signaling pathway in juvenile golden pompano (Trachinotus ovatus). A total of 450 juvenile golden pompano (9.15 ± 0.04 g) were fed three isonitrogenous diets with graded leucine levels [1.25% (control), 2.77% and 5.84%] for 8 weeks. The results showed that, compared with the control group, the WG was significantly improved in fish fed with 2.77% of dietary leucine (P < 0.05), and the 5.84% dietary leucine group had a tendency to increase. Compared to control group, 5.84% dietary leucine group significantly decreased the moisture and ash contents of whole body (P < 0.05), meanwhile, 2.77% dietary leucine group significantly decreased moisture content of whole body, but significantly improved the whole body crude lipid content (P < 0.05). Compared with the control group, the ALP level was significantly improved in fish fed with 2.77% of dietary leucine (P < 0.05). Inversely, the AST and ALT activities were significantly decreased in fish fed with 2.77% dietary leucine level (P < 0.05). Compared with the control group, GPx, T-AOC, SOD activities in group of 2.77% dietary arginine level were significantly increased (P < 0.05). However, MDA level showed a reverse trend, which was significantly decreased in fish fed with 2.77% dietary leucine level (P < 0.05). 2.77% dietary leucine levels significantly increased the relative expressions of Nrf2, HO-1, Cu/Zn-SOD, Mn-SOD and CAT (P < 0.05). In contrast, the relative expression of Keap1 showed a converse trend. Compared with the control group, the relative expressions of NF-κB, TNF-α and IL1-ß were significantly lowered in fish fed with 2.77% of dietary leucine (P < 0.05). Additionally, 2.77% dietary leucine level significantly improved the relative expressions of TGF-ß and IL-10 (P < 0.05). The 2.77% dietary leucine level significantly increased the muscular thickness compared with 5.84% dietary leucine level (P < 0.05). Furthermore, compared with the control group, the villus height and goblet cell counts were significantly improved in fish fed with 2.77% of dietary leucine (P < 0.05). These results indicated that the optimum dietary leucine plays an important role in promoting growth, enhancing antioxidant and immunity to maintain the intestinal health status of juvenile golden pompano.

Effects of dietary yeast cell wall on biochemical indices, serum and skin mucus immune responses, oxidative status and resistance against Aeromonas hydrophila in juvenile Persian sturgeon (Acipenser persicus).

The present study aims to shed light on the effects of yeast cell wall (ImmunoWall®) supplementation on biochemical indices, oxidative status, serum and mucus immune responses as well as disease resistance of juvenile Persian sturgeon (Acipenser persicus). For this purpose, one hundred fifty three juvenile Persian sturgeons (47.78 ± 0.39 g) were distributed into nine tanks (500 L) and fed with basal diets containing two levels of yeast cell wall (YCW) 0.5% (T1) and 1% (T2) and a diet without YCW as control (0%). As shown by the results obtained at the end of 56-day feeding trial, YCW had no significant effect on glucose, cortisol, SGOT, lysozyme and IgM in serum (P > 0.05) albeit an enhancement of cholesterol, LDH, ALP and SOD and ACH50 was observed in fish fed YCW supplemented diets. However, plasma triglyceride levels were lower in fish fed YCW compared with the control group. Also, total protein content, lysozyme and protease activities in skin mucus were unaffected by the supplemented diets (P > 0.05) and only total immunoglobulin and ALP enzyme activity were significantly increased in T1 and T2 groups (P > 0.05). The cumulative mortality of the fish fed supplemented diets at the end of disease challenge was 100% where cumulative mortality of those fed the control diet was 75% (P < 0.05). The present study shows that increasing immune parameters in serum and mucus of juvenile Persian sturgeon by YCW dietary supplementation did not improve resistance against Aeromonas hydrophila. According to the obtained results, the YCW supplementation at 0.5 and 1% in the juvenile Persian sturgeon diet is not recommended.

Effects of three medicinal herbs Bidens pilosa, Lonicera japonica, and Cyathula officinalis on growth and non-specific immune responses of cobia (Rachycentron canadum).

This study investigates the effects of three medicinal herbal extracts, namely Bidens pilosa (BPE), Lonicera japonica (LJE), and Cyathula officinalis (COE), on nonspecific immune parameters of cobia (Rachycentron canadum) in vitro and in vivo. During in vitro tests, BPE treatment increased reactive oxygen species (ROS) production in a dose-dependent manner in primary head kidney leukocytes. Similarly, ROS production rates were enhanced by LJE (50 and 100 mg/ml) and COE (100 mg/ml). This suggests that these three herbal extracts possess immunostimulating properties. We then conducted two feeding trials to examine the effects of these three herbal extracts on growth and innate immune parameters of cobia, and sought an optimal dietary supplementation proportion required for activating the non-specific immune responses. In the first trial, we supplemented the diet with 1, 5, or 10% of the individual extracts. After a ten-week feeding trial, no negative impacts on weight gain, feed conversion rate, and survival rate were observed in fish offered experimental diets. Further, ROS production, phagocytic capacity of the head kidney leukocytes, and serum lysozyme activity were enhanced by differing degrees in fish fed the herbal extracts compared to fish in the control group. A similar albumin/globulin ratio was seen between each experimental group and the control group regardless of the type and dose of herbal extract used, indicating these medicinal herbal extracts are safe for cobia. We then performed a 30-day feeding trial with lower extract concentrations (1, 3, and 5% of the diet) to identify dose responses in cobia at various time points so that we could establish a cost-effective manner of administering the three extracts for cobia. All BPE fed fish had higher ROS production compared to the control group, while phagocytosis rate and index were simultaneously raised in only the BPE30 group (3% BPE). Immune parameters such as ROS production, phagocytic rate, and serum lysozyme activity were triggered when fish received 30 g LJE per kg of feed. However, ROS production only increased in the LJE10 group (1% LJE) on day 30 and was not enhanced in the LJE50 group (5% LJE). Additionally, although the phagocytic rate and phagocytic index were induced in the LJE50 group, serum lysozyme activity was not elevated in this group (LJE50) at any time point examined. ROS production was greatly improved in all COE fed groups, but only the COE30 group (3% COE) showed prolonged enhanced phagocytic rate over the 30-day feeding trial.

Plant protein diet-induced hypoimmunity by affecting the spiral valve intestinal microbiota and bile acid enterohepatic circulation in Amur sturgeon (Acipenser schrenckii).

An 8-week growth trial was conducted to study enterohepatic recirculation of bile acid metabolism and the intestinal microbiota of Amur sturgeon (Acipenser schrenckii) fed with three diets, including 540 g/kg, 270 g/kg or 0 g/kg fishmeal, which was correspondingly replaced by a plant protein blend (named P0, P50 and P100, respectively). The diets were designed to be isonitrogenous, isoenergetic and essential nutrients balanced. With rising levels of dietary plant protein, disruption of the spiral valve intestinal microbiota and more morbidity with liver disease were observed in the P100 group, although there were no haematological abnormalities observed. An obvious bile acids enterohepatic circulation disorder was found with phenotypes of increased liver bile acids compensatory synthesis, and reduced expression of bile acid receptors (FXR and TGR5), which induced BA accumulative toxicity. Accompanied by increased oxidative stress, it further induced hepatic lesions and hypoimmunity, which were non-negligible reasons for the high mortality and low utilization ability of plant protein by Amur sturgeon.

Effects of dietary n-3 highly unsaturated fatty acids levels on growth, lipid metabolism and innate immunity in juvenile golden pompano (Trachinotus ovatus).

To investigate the effects of dietary n-3 highly unsaturated fatty acids (HUFA) levels on growth, lipid metabolism and innate immunity in juvenile golden pompano Trachinotus ovatus, a marine carnivorous teleost, a total of 450 fish (average body weight: 14.84 g) were randomly distributed into 18 cages at sea, each dietary group with three cages and respectively fed six diets (D1-D6) with 2.30% (D1), 0.64% (D2), 1.00% (D3), 1.24% (D4), 1.73% (D5), or 2.10% (D6) n-3 HUFA. Here, D1 with fish oil as lipid source was set as control, while D2-D6 used a mixed vegetable oil as lipid source and supplemented with docosahexaenoic acid- (DHA) and eicosapentaenoic acid- (EPA) enriched oils to adjust the n-3 HUFA levels. After 8 weeks feeding, the daily growth coefficient (DGC), specific growth rate (SGR) and feed efficiency ratio (FER) showed no significant difference among the six dietary groups (P > 0.05). The levels of EPA and DHA in serum and liver increased with the dietary n-3 HUFA levels. The activity of total superoxide disumutase (T-SOD) in serum of fish fed D4 and D5 were significantly higher than that of the other groups, whereas the opposite was true for serum IL-1ß and IL-6 levels as well as liver malondialdehyde (MDA) content. The mRNA levels of genes related to hepatic lipid metabolism including sterol regulatory element-binding protein-1 (srebp-1), fatty acid binding protein 1 (fabp1), peroxisome proliferators-activated receptor alpha (pparα), elongase of very long-chain fatty acids 5 (elovl5) and fatty acyl desaturase 2 (fads2) were down-regulated in fish fed the diets with high n-3 HUFA levels, while those of apolipoprotein b 100 (aprob 100) and carnitine palmitoyl transferase 1 (cpt1) increased significantly as increasing n-3 HUFA levels up to 1.73% (D2-D5), but decreased in the 2.10% n-3 HUFA group (D6). In addition, the expression levels of genes related to innate immunity including interleukin-10 (il-10) and transforming growth factor ß1 (tgf-ß1) increased significantly when dietary n-3 HUFA increased from 0.64% to 1.73%, whereas the opposite was true for the expression levels of nuclear factor kappa-B (nf-κb), interleukin-1ß (il-1ß), interleukin-6 (il-6) and interleukin-8 (il-8). Overall, the results indicated that dietary n-3 HUFA at 1.24-1.73% (D4-D5) can effectively improve fatty acid profiles, lipid metabolism, antioxidant capacity and immune response of golden pompano.

Probiotics, lactic acid bacteria and bacilli: interesting supplementation for aquaculture.

Probiotics administration in aquafeed is known to increase feed consumption and absorption due to their capacity to release a wide range of digestive enzymes and nutrients which can participate in digestion process and feed utilization, along with the absorption of diet components led to an increase in host's health and well-being. Furthermore, probiotics improve gut maturation, prevention of intestinal disorders, predigestion of antinutrient factors found in the feed ingredients, gut microbiota, disease resistance against pathogens and metabolism. The beneficial immune effects of probiotics are well established in finfish. However, in comparison, similar studies are less abundant in the shellfish. In this review, the discussions will mainly focus on studies reported the last 2 years. In recent studies, native probiotic bacteria were isolated and fed back to their hosts. Although beneficial effects were demonstrated, some studies showed adverse effects when treated with a high concentration. This adverse effect may be due to the imbalance of the gut microbiota caused by the replenished commensal probiotics. Probiotics revealed greatest effect on the shrimp digestive system particularly in the larval and early post-larval stages, and stimulate the production of endogenous enzymes in shrimp and contribute with improved the enzyme activities in the gut, as well as disease resistance.

Effect of EDTA enriched diets on farmed fish allergenicity and muscle quality; a proteomics approach.

Fish is one of the most common elicitors of food-allergic reactions worldwide. These reactions are triggered by the calcium-binding muscle protein ß-parvalbumin, which was shown to have reduced immunoglobulin E (IgE)-binding capacity upon calcium depletion. This work aimed to reduce gilthead seabream allergenicity using diets supplemented with a calcium chelator. Three experimental feeds were tested, differing in ethylenediaminetetraacetic acid (EDTA) supplementation, and its effects on muscle and parvalbumin's IgE-reactivity were analyzed. Chromatographic determination of EDTA showed no accumulation in the muscle and sensory results demonstrated that the lowest concentration did not affect fish quality as edible fish. Proteomics revealed one protein related to muscle contraction with significantly different relative abundance. Immunoblot assays performed with fish-allergic patients sera indicated a 50% reduction in IgE-reactivity upon EDTA presence. These preliminary results provide the basis for the further development of a non-GMO approach to modulate fish allergenicity and improve safety of aquaculture fish.

Transcriptome analysis and the effects of polyunsaturated fatty acids on the immune responses of the critically endangered angtze sturgeon (Acipenser dabryanus).

The poor understanding of nutrition needed has become a significant obstruction to artificial conservation of Yangtze sturgeon (Acipenser dabryanus) and the relationship between ployunsaturated fatty acid nutrition and the immune response of Yangtze sturgeon is remains unclear. To explore this relationship, the immune response was determined by the activities of serum immune-related enzymes and the transcriptome pattern in the spleen after feeding different fat source diets for 7 weeks. In addition, the gene expression pattern after a lipopolysaccharide (LPS) challenge was investigated in the presence of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Long-term feeding of the fish oil diets increased the serum immune-related enzyme activities, including lysozyme, acid phosphatase, and alkaline phosphatase of Yangtze sturgeon. More than 653,999 transcripts with an N50 length of 1047 bp were obtained and a final set of 280,408 unigenes was generated. After annotating the unigenes, 3549 genes were assigned to the immune system and 2839 were identified to participate in the response to the different fat sources. A transcriptome assay showed the fish oil diets moderately upregulated immune-related signaling pathways in the spleen of Yangtze sturgeon, including NLR signaling, platelet activation, Fc gamma R-mediated phagocytosis, Th17 cell differentiation, and Th1 and Th2 cell differentiation. The quantitative polymerase chain reaction (qPCR) results of candidate genes for these pathways showed similar results. The LPS challenge study revealed that DHA and EPA moderately upregulated the candidate immune-related genes and modulated excessive activation of the immune pathway by the pathogen. This study confirmed the immunomodulatory function of unsaturated fatty acids in Yangtze sturgeon. This research will provide a reference for the preparation of artificial diets for Yangtze sturgeon.

Phytochemical composition and immunobiological activity of Hawthorn Crataegus mexicana nanoencapsulated in Longfin yellowtail Seriola rivoliana leukocytes.

The aims of this study were to determine the presence of phenolic compounds in Hawthorn Crataegus mexicana, species native to Mexico, nanoencapsulated (CmNano) with maltodextrin at 100 and 170 °C (CmNano100 and CmNano170) and its antioxidant and immunological effects in Longfin yellowtail Seriola rivoliana leukocytes. The phytochemical study revealed an important level of total phenolic (TPC), flavonoid (TFC) and tannin (CTC) contents in CmNano100, which correlated with a strong antioxidant capacity. CmNano100 or 170 were safe or not cytotoxic for head-kidney (HKL) and peripheral blood (PBL) leukocytes. The in vitro study demonstrated that CmNano increased the percentage of phagocytic cells, stimulated the production of reactive oxygen species, and modulated antioxidant ability by increasing superoxide dismutase activity in leukocytes with respect to the control group. In addition, CmNano100 also increased the transcription of proinflammatory cytokine IL-1ß and down-regulated MyD88 and TNF-α mRNA transcription. These results suggest that maltodextrin nanoencapsulates protected and maintained the antioxidant properties of C. mexicana. In addition, they enhanced antioxidant and immunological parameters in Longfin yellowtail S. rivoliana leukocytes. Therefore, this study provides novel insights of CmNano for its potential application as functional food in aquaculture.

Standardization of Allergen Extracts.

Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with allergens, most allergic patients respond with production of IgE specific for several proteins/allergens in the source material. The standardization of allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting.Because the IgE binding capacity of an allergen extract is related to the content of one or a few major allergens, it is important that the standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major allergens. Owing to the complexity of allergen extracts, a key element in standardization of allergen extracts is the use of standards.This chapter describes the principles for standardization of allergen extracts to be used by research laboratories. Other chapters in this volume describe in vitro methods in detail.

Effect of sub-chronic exposure to selenium and Allium mongolicum Regel flavonoids on Channa argus: Bioaccumulation, oxidative stress, immune responses and immune-related signaling molecules.

Selenium (Se) is a micronutrient that becomes toxic when present at higher concentrations in fish tissues. Allium mongolicum Regel flavonoids (AMRF) have been documented to possess antioxidant, immunoenhancement and anti-inflammation properties. The aim of this study was to investigate the protective effects and potential mechanisms of dietary supplementation of AMRF and Se exposure on oxidative stress, immune responses and immune-related genes expression in Channa argus. A total of 480 C. argus were randomly divided into eight groups housed in twenty-four 200 L glass aquarium (3 tanks per group, 20 fish per tank). The fish were exposed for 56 days to waterborne Se at 0, 50, 100 and 200 µg/L and/or dietary AMRF at 40 mg/kg. The result indicated that AMRF exerted significant protective effects by preventing alterations in the levels of bioaccumulation, malondialdehyde, lysozyme, complement C3 and immunoglobulin M. AMRF also assists in the elevation of catalase and glutathione peroxidase in the liver and spleen while regulating the expression of immune-related genes including NF-κB p65, IκB-α, TNF-α, IL-1ß, IL-8, HSP70, HSP90, and glucocorticoid receptor after 56 days of Se exposure. Our results suggest that administration of AMRF (40 mg/kg) has the potential to combat Se toxicity in C. argus.

Effects of dietary Allium mongolicum Regel polysaccharide on growth, lipopolysaccharide-induced antioxidant responses and immune responses in Channa argus.

The present study evaluated the effects of dietary Allium mongolicum Regel polysaccharide (AMRP) on growth, lipopolysaccharide-induced antioxidant responses and immune responses in Channa argus. A basal diet was supplemented with AMRP at 0, 1, 1.5 or 2 g/kg feed for 56 days. After the 56 days feeding period, weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) were significantly increased or decreased (P < 0.05) by dietary AMRP, with the highest WG, SGR and the minimum FCR occurring in 1.5 g/kg AMRP group. Furthermore, AMRP supplementation conferred significant protective effects against LPS challenge by preventing alterations in the levels of complements 3 (C3) and complements 4 (C4), lysozyme, superoxide dismutase (SOD), glutathione-S-transferase (GST), interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) while regulating the expression of immune-related genes including heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), SOD, GST, IL-1 and TNF-α. Finally, AMRP supplementation significantly increased serum total protein, albumin and globulin concentrations and reduced mortality after LPS challenge. Taken together, our results suggest that the administration of AMRP could attenuate LPS-induced negative effects in C. argus, with 1.5 g/kg considered a suitable dose.

Increased parasite resistance of greater amberjack (Seriola dumerili Risso 1810) juveniles fed a cMOS supplemented diet is associated with upregulation of a discrete set of immune genes in mucosal tissues.

The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg-1 MOS, 2 g kg-1 cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1ß (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1ß, IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1ß whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.

Immune and Antioxidant Enzyme Response of Longfin Yellowtail (Seriola rivoliana) Juveniles to Ultra-diluted Substances Derived from Phosphorus, Silica and Pathogenic Vibrio.

BACKGROUND: This research aimed to observe the effect of homeopathic treatments prepared from Vibrio parahaemolyticus and V. alginolyticus (H1) and commercial homeopathic medication Phosphoricum acidum and Silicea terra (H2) on the immune and antioxidant response in Seriola rivoliana juveniles under usual culture conditions and challenged with V. parahaemolyticus. MATERIALS AND METHODS: Quantitative polymerase chain reaction analysis was used to study changes in the expression of key genes related to immune response, cytokines (interleukin-1ß [IL-1ß]), adapter protein for cytokine release (MyD88) and piscidin and spectrophotometric techniques to analyze the activity of antioxidant superoxide dismutase (SOD) and catalase (CAT) enzymes in Seriola rivoliana juveniles at 30 (weaning stage [WS]) and 60 (early juveniles [EJ]) days post-hatching. RESULTS: The H1 treatment led to over-expression of the IL-1ß and MyD88 genes in fish at WS and EJ with respect to control, contrary to the H2 treatment that led to under-expression of the IL-1ß, MyD88 and piscidin genes at the EJ stage. In fish challenged with V. parahaemolyticus, both H1 and H2 led to over-expression of IL-1ß and MyD88; H2 caused an over-expression of piscidin. The SOD activity was higher in H1 with respect to H2 and the control group. CAT remained relatively stable with both H1 and H2 treatments. CONCLUSIONS: The results suggest that the overall effect of H1 was due to the presence of unknown antigens in low concentrations, while the response to H2-specifically during challenge-may have been due to a stimulating effect of nano-structures, prevailing from mother tincture after sequential dilution/succussion, in a pathway similar to that attributed to nano-vaccines.