Differential polyvalent passive immune protection of egg yolk antibodies (IgY) against live and inactivated Vibrio fluvialis in fish.

Egg yolk antibodies (IgY) can be prepared in large quantities and economically, and have potential value as polyvalent passive vaccines (against multiple bacteria) in aquaculture. This study prepared live and inactivated Vibrio fluvialis IgY and immunized Carassius auratus prior to infection with V. fluvialis and Aeromonas hydrophila. The results showed that the two IgY antibodies hold effective passive protective rates against V. fluvialis and A. hydrophila in C. auratus. Further, the serum of C. auratus recognized the two bacteria in vitro, with a decrease in the bacteria content of the kidney. The phagocytic activity of C. auratus plasma was enhanced, with a decrease in the expression of inflammatory and antioxidant factors. Pathological sections showed that the kidney, spleen, and intestinal tissue structures were intact, and apoptosis and DNA damage decreased in kidney cells. Moreover, the immunoprotection conferred by the live V. fluvialis IgY was higher than that of the inactivated IgY. Addition, live V. fluvialis immunity induced IgY antibodies against outer membrane proteins of V. fluvialis were more than inactivated V. fluvialis immunity. Furthermore, heterologous immune bacteria will not cause infection, so V. fluvialis can be used to immunize chickens to obtain a large amount of IgY antibody. These findings suggest that the passive immunization effect of live bacterial IgY antibody on fish is significantly better than that of inactivated bacterial antibody, and the live V. fluvialis IgY hold potential value as polyvalent passive vaccines in aquaculture.

A leukocyte immune-type receptor specific polyclonal antibody recognizes goldfish kidney leukocytes and activates the MAPK pathway in isolated goldfish kidney neutrophil-like cells.

Leukocyte immune-type receptors (LITRs) belong to a large family of teleost immunoregulatory receptors that share phylogenetic and syntenic relationships with mammalian Fc receptor-like molecules (FCRLs). Recently, several putative stimulatory Carassius auratus (Ca)-LITR transcripts, including CaLITR3, have been identified in goldfish. CaLITR3 has four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain containing a positively charged histidine residue, and a short cytoplasmic tail region. Additionally, the calitr3 transcript is highly expressed by goldfish primary kidney neutrophils (PKNs) and macrophages (PKMs). To further investigate the immunoregulatory potential of CaLITR3 in goldfish myeloid cells, we developed and characterized a CaLITR3-epitope-specific polyclonal antibody (anti-CaL3.D1 pAb). We show that the anti-CaL3.D1 pAb stains various hematopoietic cell types within the goldfish kidney, as well as in PKNs and PKMs. Moreover, cross-linking of the anti-CaL3.D1-pAb on PKN membranes induces phosphorylation of p38 and ERK1/2, critical components of the MAPK pathway involved in controlling a wide variety of innate immune effector responses such as NETosis, respiratory burst, and cytokine release. These findings support the stimulatory potential of CaLITR3 proteins as activators of fish granulocytes and pave the way for a more in-depth examination of the immunoregulatory functions of CaLITRs in goldfish myeloid cells.

Characterization of CD83 homologs differently expressed during monocytes differentiation in ginbuna crucian carp, Carassius auratus langsdorfii.

CD83 is a costimulatory molecule of antigen-presenting cells (APCs) that plays an important role in eliciting adaptive responses. It is also a well-known surface protein on mature dendritic cells (DCs). Furthermore, monocytes have been reported to differentiate into macrophages and monocyte-derived dendritic cells, which play an important role in innate immunity. CD83 expression affects the activation and maturation of DCs and stimulates cell-mediated immune responses. This study aims to reveal the CD83 expression during monocyte differentiation in teleosts, and the CD83 homologs evolutionary relationship. This study found two distinct CD83 homologs (GbCD83 and GbCD83-L) in ginbuna crucian carp (Gb) and investigated the evolutionary relationship among GbCD83 homologs and other vertebrates and the gene and protein expression levels of the homologs during 4 days of monocyte culture. The phylogenetic tree showed that the two GbCD83 homologs are classified into two distinct branches. Interestingly, only ostariophysians (Gb, common carp, rohu, fathead minnow and channel catfish), but not neoteleosts, mammals, and others, have two CD83 homologs. Morphological observation and colony-stimulating factor-1 receptor (CSF-1R), CD83, CD80/86, and CCR7 gene expressions illustrated that there is a differentiation of monocytes isolated from peripheral blood leukocytes after 4 days. Specifically, gene expression and immunocytochemistry revealed that GbCD83 is mainly expressed on monocytes at the early stage of cell culture, whereas GbCD83-L is expressed in the latter stage. These findings provided the first evidence of differential expression of CD83 homologs during monocytes differentiation in teleost.

Investigation of putative antimicrobial peptides in Carassius gibel, revealing a practical approach to screening antimicrobials.

Antimicrobial peptides (AMPs) and their mimics are rapidly gaining attention as a new class of antimicrobials due to their clinical potential. AMPs are widely distributed throughout nature and participate in the innate host defense. In this study, 18 AMPs, including 3 ß-defensins, 3 hepcidins, 4 liver-expressed antimicrobial peptide 2 (LEAP-2) compounds, 4 g-type lysozymes, 2 c-type lysozymes, and 2 NK-lysins, were identified from the genome of Carassius auratus by a homologous search and were further classified based on their fundamental structural features and molecular phylogeny. C. auratus AMPs were found to be ubiquitously distributed in all tested tissues and showed similar expression profiles, with the exception of ß-defensins, when RT-qPCR was used to investigate the tissue distribution of AMPs in healthy Carassius gibel. In addition, the expression levels of NK-lysin genes in the tested tissues tended to be upregulated upon bacterial and viral infection when representative NK-lysins were chosen to examine their relative expression levels in various tissues. Importantly, the synthetic peptide caNKL2102-119, which targets the functional domain of saposin B in caNK-lysins, could effectively counter Aeromonas hydrophila, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration (MIC) values of 3-6 µg/mL, as well as inhibit the proliferation of spring viraemia of carp virus (SVCV). These results provide potential targets for antibiotic-free breeding in the aquaculture industry.

Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract.

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg-1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Induction of both local and systemic immunity by in vivo injection of PHA into ginbuna carp fin.

Phytohemagglutinin (PHA) is a well-known mitogen inducing activation and proliferation of lymphocytes, particularly T lymphocytes in vitro. PHA has also been used in vivo for assessing cell-mediated immunity in non-mammalian vertebrates, particularly in birds. However, it has been suggested that local inflammation as a direct result of tissue damage could be responsible for skin swelling after PHA injection, in addition to induction of T lymphocyte mitogenesis. In order to understand the complex nature of this response in fish we investigated the accumulation of cell types chronologically in dorsal fin of ginbuna crucian carp Carassius auratus langsdorfii after PHA injection. Neutrophils appeared first and showed a peak response on day 1, decreasing gradually and followed by macrophages and blast cells while lymphocytes increased later with a peak response on day 5. The number of accumulated cells was significantly higher in PHA-injected fish than controls in most cases. Lymphocytes identified as CD4-1+and CD8α+ were significantly more abundant in PHA-injected fish than in control fish throughout the 7-day experimental period except on day 1, while the number of IgM+ lymphocytes was higher in PHA-injected fish only on day 1. In the blast cell fraction, the number of CD4-1+ lymphocytes was significantly higher in PHA-injected fish than in control fish throughout experimental period, except on day 1. We also document the migration of neutrophils from the kidney to the fin through blood, followed by granulopoiesis in the kidney. These results suggest that adaptive immunity as well as innate immunity was induced by in vivo stimulation with PHA.

A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies.

In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

Effects of mixed antimicrobial peptide on the growth performance, antioxidant and immune responses and disease resistance of Pengze crucian carp (Carassius auratus var. Pengze).

Antimicrobial peptides have broad-spectrum antibacterial properties and low drug resistance, and they demonstrate great potential as antibiotic substitutes. In this study, five dietary mixed antimicrobial peptide supplement groups were set and fed to Pengze crucian carp for 10 weeks. The 6 groups were G0 (control group) and 5 additional groups: G1 (100 mg/kg), G2 (200 mg/kg), G3 (400 mg/kg), G4 (800 mg/kg) and G5 (1600 mg/kg). The results showed that the final body weight (FBW), weight gain rate (WGR) and specific growth rate (SGR) of fish in G1 and G2 were higher than those of fish in the control group, and G1 was significantly higher than G0 (P < 0.05). In addition, the FBW, WGR, and SGR of the G3 group were significantly lower than those of the G0 group. The chymotrypsin, lipase and amylase activities of G1 and G2 were significantly upregulated compared with G0 and reached peak values in G1. The activity of T-AOC and SOD in the addition group was higher (except G2 and G4) than that in the control groups, and significantly increased in G3 compared to the control group. The activity of MDA in the addition group was lower than that in the control group (p > 0.05). The expression levels of TLR-4, MYD88 and TNF-α in the three organs of the addition group were higher than those in G0 and reached the peak value in G3 (p < 0.05). Furthermore, the expression levels of TLR-4, MYD88 and TNF-α in the three organs of G3 were significantly lower than those in G0 and lower than those in the other supplemented groups. The expression levels of IL-10 and IL-11 tended to be upregulated after A. hydrophila challenge, and G3 in different organs was significantly higher than that in other supplemented groups and G0. The results of this study show that an appropriate amount of mixed antimicrobial peptides can improve the growth performance and antioxidant and immune capabilities of Pengze crucian carp and can also play a positive role in the treatment of A. hydrophila infection.

Molecular characterization and biological effect of a C-type lectin receptor in Qihe crucian carp, Carassius auratus.

C-type lectin receptors, as the important members of pattern-recognition receptors, play the crucial roles in the innate immune system, which discriminate self and non-self by recognizing and binding the carbohydrates on the surface of microorganism. In this study, we identified a C-type lectin receptor gene in Qihe crucian carp Carassius auratus (named as CaCLR). The full-length cDNA of CaCLR was composed of 1130 bp, with a 226 bp 5'-untranslated region (UTR), a 792 bp ORF encoding a 263aa protein, and a 112 bp 3'-UTR with a polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of CaCLR is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. With regard to the mRNA transcript of CaCLR, it was ubiquitously detected in the tested tissues, among which it was the most abundant in head kidney. The temporal expressions of CaCLR were obviously up-regulated in liver, spleen, kidney, and head kidney after Aeromonas hydrophila and poly I: C challenge, respectively, and the patterns of expression changes were in a time-depended manner. The recombinant CaCLR (rCaCLR) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with lipopolysaccharide (LPS), peptidoglycan (PGN), ß-Glucan, and Mannan, as well as five microorganisms including fungus (Saccharomyces cerevisiae), Gram-negative bacteria (A. hydrophila, E. coli and Vibrio anguillarum), and Gram-positive bacteria (Micrococcus lysodeikticus). In the presence of rCaCLR, the eliminating capacity against A. hydrophila could be enhanced in C. auratus. Taken together, CaCLR is involved in the antibacterial defense in C. auratus.

Expression and regulation of ccBAX by miR-124 in the caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

Bcl2 family proteins play a critical role in cell death or survival. BAX, the death-promoting protein of bcl2 family, mediated mitochondrial pathway inducing cells' apoptosis in mammal. MiRNAs have been implicated as negative regulators down-regulating genes' expression after post-transcriptional level. At present, little is known about the regulatory mechanism of miRNA on the Bcl2 family proteins during CyHV-2 infection in silver crucian carp (Carassius auratus gibelio). In this study, the ccBAX (silver crucian carp BAX) gene was cloned and expressed, and polyclonal antibodies were raised in mouse against the purified ccBAX-GST fusion protein. The structure analysis indicated that ccBAX protein included four conserve domains (BH1, BH2, BH3 and transmembrane domains) and the expression of ccBAX protein occurred throughout the cells. Furthermore, two miRNAs (miR-124 and miRNA-29b) were identified to negatively regulate ccBAX gene expression in GiCF cell. miR-124 was found to suppress the expression of WT-ccBAX (wild type), but not the MT-ccBAX (mutant). Overall, the results demonstrated that the expression of the ccBAX gene was significantly down-regulated by miR-124 in silver crucian carp (Carassius auratus gibelio) during CyHV-2 infection.

Innate cell-mediated cytotoxicity of CD8+ T cells against the protozoan parasite Ichthyophthirius multifiliis in the ginbuna crucian carp, Carassius auratus langsdorfii.

Cytotoxic T cells are known to have the ability to kill microbe-infected host cells, which makes them essential in the adaptive immunity processes of various vertebrates. In this study, we demonstrated innate cell-mediated cytotoxicity of CD8+ T cells against protozoan parasites found in the ginbuna crucian carp. When isolated effector cells such as CD8+, CD4+ (CD4-1+), or CD8- CD4- (double-negative, DN), from naïve ginbuna crucian carp were co-incubated with target parasites (Ichthyophthirius multifiliis), CD8+ cells from the kidney and gill showed the highest cytotoxic activity. On the other hand, DN cells, which include macrophages and CD4- CD8- lymphocytes, showed the lowest cytotoxic activity against I. multifiliis. Additionally, the cytotoxic activity of CD8+ cells was found to significantly decrease in the presence of a membrane separating the effector cells from I. multifiliis. Furthermore, the serine protease inhibitor 3,4-dichloroisocoumarin and perforin inhibitor concanamycin A significantly inhibited the cytotoxic activity of CD8+ cells. These results demonstrate that CD8+ T cells of ginbuna crucian carp can kill extracellular parasites in a contact-dependent manner via serine proteases and perforin. Therefore, we conclude that CD8+ T cells play an essential role in anti-parasite innate immunity of teleost fish.

Stimulatory effect of dietary chasteberry (Vitex agnus-castus) extract on immunity, some immune-related gene expression, and resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus).

Chasteberry is highly recommended as an herbal medicine across the globe for treating of many gynaecological disorders. In this study, chasteberry extract (CBE) was supplemented in goldfish diet to evaluate the immunity responses at the cellular and molecular levels. Moreover, after the feeding trial, the fish were challenged with Aeromonas hydrophila. The fish (300 individuals, 2.4 ± 0.12 g initial weight) were randomly distributed in 12 tanks and were fed with 0 (control), 5, 10, and 15 g CBE per kg of feed for 8 weeks. Based on the results, lysozyme activity, alkaline phosphatase, and total immunoglobulin (Ig) in the skin mucus samples were significantly enhanced in the fish fed with 15 g/kg CBE (P < 0.05). Moreover, dietary CBE positively affected lysozyme activity, complement components, and IgM content of the serum samples compared to the control group. Also, the number of monocytes and lymphocytes were increased significantly with increasing CBE in the diet (P < 0.05). The highest mRNA levels of tumor necrosis factors (TNF-α, TNF-2α) and Lysozyme were observed in 15 g/kg CBE treatment. After the challenge test, the highest relative percentage survival value (60%) was observed in the fish fed with 15 g/kg CBE. We concluded that dietary CBE especially at 15 g/kg has an immunomodulatory effect in goldfish by stimulating the innate immunity and some inflammatory cytokines as well as disease resistance against A. hydrophila.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification.

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

Effects of dietary fish meal replacement by fermented moringa (Moringa oleifera Lam.) leaves on growth performance, nonspecific immunity and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III).

This study was aimed to evaluate the effects of partial replacement of fish meal by fermented moringa leaves (FMLs) on growth performance, serum biochemistry, antioxidant status, nonspecific immunity, and resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III). Four isonitrogenous and isoenergetic balanced diets, including three FML diets (substituting 20%, 40%, 60% of the fish meal in basal diet, F20, F40 and F60, respectively) and a basal diet (a diet containing 10% fish meal) were used. Each diet was randomly allocated to four fish groups (F20, F40, F60 and control) reared in a recirculating system. After 50 days of the feeding trial, fish were challenged by A. hydrophila. The result revealed that final mean body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) were significantly increased (P < 0.05) in F20 and F40 groups compared with the control group. Decreased hepatosomatic index (HSI), body crude lipid, serum aspartate transaminase (AST) and serum alanine aminotransferase (ALT) activities, and increased serum alkaline phosphatase (AKP) and serum glutathione peroxidase (GPx) activities were observed in F40 and F60 groups compared with the control and F20 groups. All FMLs-supplemented groups increased (P < 0.05) serum superoxide dismutase (SOD), catalase (CAT) and lysozyme activities, complement component 3 (C3) and serum immunoglobulin M (IgM) concentration, or decreased serum malondialdehyde (MDA) and protein carbonyl (PCC) contents (P < 0.05). After the challenge test, the significant downregulation of toll-like receptors2 (TLR2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-8 mRNA transcription levels was observed in spleens of FMLs supplemented groups. Dietary F40 and F60 showed higher (P < 0.05) relative percent survival (RPS) (48.72% and 43.59%, respectively) against A. hydrophila infection than control. These results indicate that, as a dietary fish meal substitute, FMLs enhance the growth, and antioxidant and immune response, and regulate the expression of immune-related genes and increase disease resistance against A. hydrophila via TLR2 pathway in gibel carp, with greatest effects of 40% fish meal substitution.

Transcriptome analysis provides insights into the antiviral response in the spleen of gibel carp (Carassius auratus gibelio) after poly I: C treatment.

Gibel carp (Carassius auratus gibelio) is an important commercial fish that has become one of the most cultured fishes in the region of Yangtze River in China. However, the fish faces increasing hazard due to cyprinid herpesvirus 2 (CyHV-2) infection, which has caused great economic losses. In this study, healthy gibel carp were intraperitoneally injected with different doses of poly I:C at 24 h before CyHV-2 challenge. Results showed that the mortality decreased and peak death time appeared later in the fish injected with poly I:C at a dose of 10 µg/g body weight. To explore what gene plays an important role after poly I:C treatment, the transcriptome analysis of the gibel carp spleen was further performed. Compared with the PBS group, 1286 differentially expressed genes (DEGs) were obtained in the poly I:C-treated fish, including 1006 up-regulated and 280 down-regulated DEGs. GO analysis revealed that the most enriched DEGs responded to "biological regulation", "regulation of cellular process" and "regulation of biological process". Meanwhile, KEGG enrichment analysis showed that the DEGs were mainly mapped on the immune pathways like "TNF signal pathway", "p53 signal pathway" and "JAK-STAT signal pathway", suggesting that these signal pathways may be responsible for the delayed peak of CyHV-2 infection in gibel carp after poly I:C treatment. Taken together, this study provides insights into the immune protection effect of poly I:C against CyHV-2 infection, as well as providing useful information for antiviral defense in gibel carp.

Improvement of non-specific immunity, growth, and activity of digestive enzymes in Carassius auratus as a result of apple cider vinegar administration to diet.

This study was conducted to evaluate the effects of apple cider vinegar (ACV) administration on non-specific immunity of serum and skin mucus, growth indices, and activity of digestive enzymes (amylase, lipase, and protease) in Carassius auratus. For this purpose, 180 fish (weighing 7.35 ± 0.19 g) were allocated to 4 treatment groups with 3 replications in a completely randomized design. Fish were fed for 105 days using a basal diet supplemented with 0% (control), 1% (T 1), 2% (T 2), and 4% (T 3) ACV (contained 5% acetic acid). Results showed a significant increase in lysozyme activity, ACH50, and total immunoglobulin of skin mucus in fish fed with T2 diet (p < 0.05). Total immunoglobulin and lysozyme activity were significantly lower in the serum of fish fed with control diet than those fed with the mentioned treatment (p < 0.05). The highest value was observed in fish fed with T2 diet. Minimum (p < 0.05) complement activity (1.52 ± 0. 25 U ml-1) was observed in fish fed with control diet. The mean of the final weights (17.35 ± 1.39 g), daily growth (1.0 ± 0.01 g), and specific growth rate (2.19 ± 0.14) was significantly higher in T3 diet group than the controls (p < 0.05). While the highest amylase-specific activity was observed in the controls (p < 0.05), there was a significant increase in specific activity of protease, lipase, and alkaline phosphatase in T2 diet group (p < 0.05). According to the results of this study, the inclusion of a limited quantity of ACV (4%) into the diet can improve immunity and growth parameters in C. auratus.

Dietary supplementation of Geotrichum candidum improves growth, gut microbiota, immune-related gene expression and disease resistance in gibel carp CAS â ¢ (Carassius auratus gibelio).

Geotrichum candidum is a filamentous fungus mostly used in the dairy industry for cheese ripening and flavoring. The current study was designed to evaluate the potential probiotics effect of dietary supplementation of G. candidum on growth, digestive enzymes activities, gut microbiota and immune-related gene expression and disease resistance in Gibel carp CAS Ⅲ (Carassius auratus gibelio) against A. hydrophila infections. For this purpose, 420 healthy fish (initial body weight: 7.09 ± 0.02g) were randomly divided into 7 feeding groups in triplicates of 20 fish in each tank. Seven isocaloric and isonitrogenous diets were prepared, supplemented with 6 different doses of G. candidum viz; C: 0, T1:106, T2:107, T3:108, T4:109, T5:1010, T6:1011 CFU/kg diet and fed to fish twice a day for 60 days. Results showed significantly higher weight gain (WG) and feed efficiency (FE) in groups fed 106 and 108 CFU/kg diet compared to the control (P < 0.05). All G. candidum fed groups showed higher intestinal α-amylase activity (P < 0.05) while no difference in lipase and trypsin activity compared to the control group. Before challenge test, gut microbial diversity analysis revealed relatively more abundance of bacteria belonging to phylum Proteobacteria, Actinobacteria, Firmicutes and Bacteriodetes in group fed G. candidum supplemented diet and higher percentage of pathogenic bacteria belonging to order Aeromonadales and Vibrionales in control post-challenge. After challenge test immune response expressed numerically higher survival rate (P > 0.05) and significantly higher (P < 0.05) Respiratory Burst Activity(RBA), Immunoglobulin M level (IgM), Aspartate Transaminase activity (AST) and improved (P > 0.05) lysozyme activity, and Alanine Transaminase activity (ALT) in groups fed 106 and 108 CFU/kg G. candidum supplemented diet. The expression of immune related gene (IL-I ß, TNF- α, HSP70 and TLR-2) in liver of the fish were significantly affected (P < 0.05) by supplementation of G. candidum post bacterial challenge. The results showed that G. candidum is a potential probiotic as it could improve feed utilization, immunity and cause no harmful effects on growth of gibel carp at higher supplementation levels. The productive results can be achieved by using the lower dose as 106-108 CFU/kg diet.

ß-glucan and anisodamine can enhance the immersion immune efficacy of inactivated cyprinid herpesvirus 2 vaccine in Carassius auratus gibelio.

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a ß-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, ß-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that ß-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, ß-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in ß-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of ß-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Modulation of the growth performance, body composition and nonspecific immunity of crucian carp Carassius auratus upon Enteromorpha prolifera polysaccharide.

The present study investigated dietary Enteromorpha prolifera polysaccharide (EPP) on the growth performance, body composition and non-specific immunity of crucian carp Carassius auratus. Crucian carps with body weight of 51.24 ± 4.08 g were randomly divided into five groups: one group was fed with basic diet, while the other four groups were fed with diets containing 20, 40, 60 and 80 g/kg EPP. After 60 days of feeding, dietary administration of 40 g/kg EPP increased the body weight gain rate, feed conversion ratio, specific growth rate, body crude protein content, intestine digestive enzyme activities, serum lysozyme activity, acid phosphatase activity, alkaline phosphatase activity, complement 3 level, superoxide dismutase activity and catalase activity and decreased body fat content and serum total cholesterol, triacylglycerol, alanine aminotransferase activity and glutamic oxaloacetic transaminase activity compared with those of the control group. All levels of EPP improved the resistance to Aeromonas hydrophilia compared with the control group. The results indicated that EPP could promote the growth of crucian carp and improve their disease resistance and may be used as a dietary supplement for crucian carp.

Study on Immune Response of Organs of Epinephelus coioides and Carassius auratus After Immersion Vaccination With Inactivated Vibrio harveyi Vaccine.

Immersion vaccination relies on the response of fish mucosa-associated lymphoid tissues, the Crucian carp (Carassius auratus) and Grouper (Epinephelus coioides) were researched in this paper to examine local mucosal immune responses and associated humoral system responses following immersion vaccination. We administered 1.5 × 107 CFU/ml formalin-inactivated Vibrio harveyi cells and measured mucus and serum antibody titers as well as IgM, MHC II mRNA levels in immune organs. The mucosal antibody response preceded the serum response indicating a role for local mucosal immunity in immersion vaccination. IgM and MHC II mRNA levels were relatively greater for the spleen and head kidney indicating the importance and central position of systemic immunity. Expression levels were also high for the gills while skin levels were the lowest. IgM and MHC II mRNA levels were altered over time following vaccination and the hindgut, liver and spleen were similar indicating a close relationship, so the absolute value of r is used to analyze the correlation among different organs immunized. It can be inferred the existence of an internal immune molecular mechanism for Immune synergy hindgut-liver-spleen, from the peak time (14th day), the relative ratio of genes expression in the same tissues between the immunized grouper and the control group (26 times), and Pearson correlation coefficient (0.8<|r|<1). Injection challenges with live V. harveyi indicated that the relative protection rates for the crucian carp and Grouper was basically the same at 44.4% and 47.4%, respectively. It is believe that crucian carp may be used as a substitute for the valuable grouper in immunity experiment, just from aspect of the relative percent survival (RPS) and how it changes with time. But they were not consistent about the IgM mRNA expression between that of crucian carp and grouper after immersion the Vibrio vaccine.