Alteration of lipid metabolism, autophagy, apoptosis and immune response in the liver of common carp (Cyprinus carpio) after long-term exposure to bisphenol A.

Bisphenol A (BPA), as a phenolic compound, is harmful to human health, and its residue in the aquatic environment also threatens the health of aquatic animals. In this research, the toxicity effects of BPA on liver tissues were evaluated in common carp (Cyprinus carpio) after long-term exposure. Fish were exposed to five concentrations of BPA (0, 0.01, 0.1, 0.5 and 2 mg/L) for 30 days. The blood and liver tissues were gathered to analyze biochemical indices and genes transcription levels. The data related to lipid metabolism showed that BPA exposure increased serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels, upregulated the expressions of fatp1, pparγ, fas, atgl, hsl, pparα, cpt1b, acox-1, and downregulated the expression of dgat1 in liver. Antioxidative parameters displayed a reduced antioxidant ability and increased lipid peroxidation after BPA exposure. Meanwhile, the upregulations of nrf2, ho-1, cyp1a and cyp1b genes revealed an adaptive response mechanism against oxidative stress-induced adverse effects. After 30 days of exposure, BPA induced apoptosis and endoplasmic reticulum stress (ERS) via upregulating the expression levels of apoptosis and ERS-related genes and increasing Ca2+ concentration in liver. Moreover, the downregulation of mtor and the upregulation of atg3, atg7, tfeb, uvrag and mcoln1 indicated that BPA could influence the normal process of autophagy. Furthermore, BPA exposure activated toll like receptors (TLRs) pathway to mediate the inflammatory response. Our results demonstrated that BPA exposure disturbed lipid metabolism, and induced oxidative stress, ERS, apoptosis, autophagy and inflammatory response in the liver of common carp. These findings contributed to the understanding of hepatotoxicity mechanism induced by BPA in fish.

Effects of high-fat diet on antioxidative status, apoptosis and inflammation in liver of tilapia (Oreochromis niloticus) via Nrf2, TLRs and JNK pathways.

Fatty liver injury (or disease) is a common disease in farmed fish, but its pathogenic mechanism is not fully understood. Therefore the present study aims to investigate high-fat diet (HFD)-induced liver injury and explore the underlying mechanism in fish. The tilapia were fed on control diet and HFD for 90 days, and then the blood and liver tissues were collected to determine biochemical parameter, gene expression and protein level. The results showed that HFD feeding signally increased the levels of plasma aminotransferases and pro-inflammatory factors after 60 days. In liver and plasma, HFD feeding significantly suppressed antioxidant ability, but enhanced lipid peroxidation formation, protein oxidation and DNA damage after 60 or 90 days. Further, the Nrf2 pathway and antioxidative function-related genes were adversely changed in liver of HFD-fed tilapia after 60 and/or 90 days. Meanwhile, HFD treatment induced apoptosis via initiating mitochondrial pathway in liver after 90 days. Furthermore, after 90 days of feeding, the expression of genes or proteins related to JNK pathway and TLRs-Myd88-NF-κB pathway was clearly upregulated in HFD treatment. Similarly, the mRNA levels of inflammatory factors including tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8 and IL-10 were also upregulated in liver of HFD-fed tilapia after 60 and/or 90 days. In conclusion, the current study suggested that HFD feeding impaired antioxidant defense system, induced apoptosis, enhanced inflammation and led to liver injury. The adverse influences of HFD in the liver might be due to the variation of Nrf2, JNK and TLRs-Myd88-NF-κB signaling pathways.

Oxidative stress, ion concentration change and immune response in gills of common carp (Cyprinus carpio) under long-term exposure to bisphenol A.

Bisphenol A (BPA) is a well-known phenolic environmental estrogen, widely distributed in the aquatic environment, which poses a toxic risk to the health of aquatic organisms. This study aimed to assess the effect of BPA on common carp gills by analyzing oxidative stress, ion equilibrium and immune response. Fish were exposed to five concentrations of BPA (0, 0.01, 0.1, 0.5, and 2 mg/L) for 30 days. Then gills were collected to assay biochemical parameters and gene expression. The results showed that BPA could decrease the levels of total antioxidant capacity (T-AOC), catalase (CAT), glutathione (GSH) and glutathione S-transferase (GST) and increase the levels of superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxy-2 deoxyguanosine (8-OHdG). The gene expression showed that BPA (2 mg/L) could affect the nuclear erythroid 2-related factor 2 (nrf2) signaling pathway, upregulate the gene expression of nrf2 and heme oxygenase 1 (ho-1). Meanwhile, BPA was found to change the activity of Na+/K+ ATPase, and increased the concentrations of Na+ and Ca2+ in gills of common carp. Also, high BPA concentration (0.5 or 2 mg/L) exposure increased the activity of alkaline phosphatase (AKP), blocked mRNA level of lysozyme-c (c-lyz), activated Toll-like receptors (TLRs) signaling pathway, enhanced the mRNA levels of toll-like receptor 2 (tlr2), receptor 4 (tlr4), myeloid differentiation factor 88 (myd88), interferon regulatory factor 3 (irf3), interleukin 1ß (il-1ß), interleukin 6 (il-6) and interleukin 10 (il-10). Overall, these results suggested that high BPA could induce oxidative damage, ion imbalance, immunosuppression and inflammatory response in gills of common carp.

Anti-oxidative, anti-inflammatory and hepatoprotective effects of Radix Bupleuri extract against oxidative damage in tilapia (Oreochromis niloticus) via Nrf2 and TLRs signaling pathway.

Radix Bupleuri extract (RBE) is one of the most popular oriental herbal medicines, which has anti-oxidative and anti-inflammatory properties. However, its protective effects and underlying molecular mechanisms on oxidative damage in tilapia are still unclear. The aims of the study were to explore the anti-oxidative, anti-inflammatory and hepatoprotective effects of RBE against oxidative damage, and to elucidate underlying molecular mechanisms in fish. Tilapia received diet containing three doses of RBE (0, 1 and 3 g/kg diet) for 60 days, and then were given an intraperitoneal injection of H2O2 or saline. Before injection, RBE treatments improved growth performance and partial anti-oxidative capacity in tilapia. After oxidative damage, RBE pretreatments were able to signally reduce the higher serum aminotransferases, alkaline phosphatase (AKP) and liver necrosis. In serum and liver, the abnormal lipid peroxidation level and antioxidant status induced by H2O2 injection were restored by RBE treatments. Furthermore, RBE treatments activated erythroid 2-related factor 2 (Nrf2) signaling pathway, which promoted the gene expression of heme oxygenase 1 (HO-1), NAD(P) H:quinone oxidoreductase 1 (NQO-1), glutathione-S-transferase (GST) and catalase (CAT). Meanwhile, RBE treatments reduced inflammatory response by inhibiting TLRs-MyD88-NF-κB signaling pathway, accompanied by the lower interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-8 mRNA levels. In addition, RBE treatments upregulated complement (C3) gene expression and downregulated heat shock protein (HSP70) gene expression. In conclusion, the current study suggested that RBE pretreatments protected against H2O2-induced oxidative damage in tilapia. The beneficial activity of RBE may be due to the modulation of Nrf2/ARE and TLRs-Myd88-NF-κB signaling pathway.

Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed.

The aim of this research was to improve nutritive value of fishmeal-based feed by lactobacilli in order to achieve satisfactory nutrient availability needed to support fish development. Feed was solid-state treated at a laboratory scale with the combination of Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 in different experimental settings, which included the variation of strain ratio, total lactobacilli concentration, percentage of moisture and duration of incubation. Short peptides, soluble proteins, phospho-, neutral and unsaturated lipids were quantified. Differences among treated and control feeds were evaluated by Student t-test, while Gaussian process regression (GPR) modeling was employed to simulate the incubation process and define the optimal treatment combination in the context of overall feed nutritional profile. Treatment duration was shown to be the critical determinant of final outcome, either as single factor or via interaction with strain ratio. Optimal nutrient balance was achieved with 12 h incubation period, 260% moisture, 75:25 and 50:50 BGHN14:BGT10 ratios and 200 mg of lactobacilli per g of dry feed. This study should serve as the basis for large-scale tests which would simulate on-farm production of both fishmeal-based and unconventional, lower cost aquafeed with added value.

Antioxidative, anti-inflammatory and hepatoprotective effects of resveratrol on oxidative stress-induced liver damage in tilapia (Oreochromis niloticus).

Resveratrol, a dietary polyphenol, has been shown to exert antioxidation, hepatoprotection, anti-inflammation and immunostimulation. However, the effects and underlying mechanism of resveratrol on liver injury in fish are still unclear. In the present study, we investigated the potential protective effects and mechanism of resveratrol on oxidative stress-induced liver damage in tilapia. Fish were fed diet containing four doses of resveratrol (0, 0.1, 0.3, and 0.6 g/kg diet) for 60 days, and then given an intraperitoneal injection of H2O2 or saline. The results showed that administration of resveratrol significantly ameliorated H2O2-induced liver injury. In serum and liver, resveratrol treatment suppressed the oxidative stress, as evidenced by the decline of lipid peroxidation level and increase of antioxidant activity. Resveratrol also activated erythroid 2-related factor 2 (Nrf2) signaling pathway and enhanced the heme oxygenase 1 (HO-1), NAD(P) H:quinone oxidoreductase 1 (NQO-1), glutathione S-transferase (GST) mRNA levels. Meanwhile, resveratrol treatment repressed TLR2-Myd88-NF-κB signaling pathway to decrease the inflammatory response in H2O2-induced liver injury as evidenced by the lower interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-8 mRNA levels and higher IL-10 mRNA level. Moreover, resveratrol treatment attenuated immunotoxicity in liver of H2O2-treated fish, accompanied by upregulation of hepcidin (HEP), complement 3 (C3) and lysozyme (LZM) mRNA levels. Overall results suggested that the protection of resveratrol on H2O2-induced liver injury, inflammation and immunotoxicity was due to its antioxidant property and its ability to modulate the Nrf2 and TLR2-Myd88-NF-κB signaling pathways.

Antioxidative, inflammatory and immune responses in hydrogen peroxide-induced liver injury of tilapia (GIFT, Oreochromis niloticus).

Oxidative stress has been implicated in the pathogenesis of many liver diseases in fish, but the molecular mechanism is still obscure. Here, we used hydrogen peroxide (H2O2) as a reactive oxygen species (ROS) to induce liver injury and assess underlying molecular mechanism linking oxidative stress and liver injury in fish. Tilapia were injected with various concentrations of H2O2 (0, 40, 120, 200, 300 and 400 mM) for 72 h. The blood and liver were collected to assay biochemical parameters and genes expression after 24, 48 and 72 h of injection. The results showed that treatments with higher H2O2 levels (300 and/or 400 mM) significantly increased the levels of GPT, GOT, AKP and MDA, and apparently decreased the levels of TP, ALB, SOD, GSH, CAT, GST and T-AOC throughout of the 72 h. The gene expression data showed that treatments with 200, 300 and/or 400 H2O2 suppressed Nrf2/keap1 pathway and its downstream genes including ho-1, nqo1 and gsta, activated inflammatory response via enhancing the mRNA levels of nf-κb, tnf-α, il-1ß and il-8, and attenuating il-10 mRNA level, and caused immunotoxicity through downregulating the genes expression of c3, hep, lzm and Igm for 24, 48 and/or 72 h. Additionally, there was a mild or strong increase in levels of nrf2 and its subsequent antioxidant genes or enzymes such as ho-1, nqo1, gst, CAT and SOD in treatments with lower concentrations of H2O2 (40 or 120 mM) for 24 and/or 48 h. Overall results suggested that H2O2 hepatotoxicity was mainly concerned with lipid peroxidation, impairment antioxidant defense systems, inflammatory response and immunotoxicity, and Nrf2/Keap1 and NF-κB signaling pathways played important roles in oxidative stress-induced liver injury in fish.

Supplementation of lactobacilli improves growth, regulates microbiota composition and suppresses skeletal anomalies in juvenile pike-perch (Sander lucioperca) reared in recirculating aquaculture system (RAS): A pilot study.

This research aimed to test the effects of lactobacilli, applied to cultured pike-perch, either through hydrolyzed OTOHIME fish diet, or through Artemia nauplii, on fish growth, microbiota balance and skeletal development. On the 12th Day Post Hatching (DPH) fish were divided into following treatment groups: two groups received the combination of OTOHIME and nauplii enriched either with Lactobacillus paracasei BGHN14+Lactobacillus rhamnosus BGT10 or with Lactobacillus reuteri BGGO6-55+Lactobacillus salivarius BGHO1, and one group received OTOHIME hydrolyzed by BGHN14+BGT10 and non-enriched nauplii. Control group received non-enriched nauplii and non-hydrolyzed OTOHIME. The treatment lasted 14days and fish were sacrificed on the 26th DPH for the assessment of digestive enzyme activity and microbiota composition. Individual total lengths and individual body weights were recorded at the end of the treatments, on the 26th DPH, and also on the 45th DPH, in parallel with the evaluation of skeletal deformities and fish survival. Our results indicated positive effect of Artemia enriched with BGGO6-55+BGHO1 on fish growth, skeletal development and trypsin to chymotrypsin activity ratio (T/C), as an indicator of protein digestibility. Hydrolysis of OTOHIME was also associated with better skeletal development, higher T/C values and lower levels of Aeromonas and Mycobacterium spp., which are important fish pathogens. Though additional testing in larger cohort studies is needed, these observations are promising in terms of usage of probiotics for improved environmentally friendly production of pike-perch in Recirculating Aquaculture System (RAS).

Protective effect of Ganoderma lucidum polysaccharide against carbon tetrachloride-induced hepatic damage in precision-cut carp liver slices.

The aim of the present study was to investigate the protective effects of Ganoderma lucidum polysaccharide (GLPS) against carbon tetrachloride (CCl4)-induced hepatotoxicity in vitro in common carp. Precision-cut liver slices (PCLSs), which closely resemble the organ from which they are derived, were employed as an in vitro model system. GLPS (0.1, 0.3, and 0.6 mg/ml) was added to PCLS culture system before the exposure to 12 mM CCl4. The supernatants and slices were collected to detect molecular and biochemical responses to CCl4 and PCLS treatments. The levels of CYP1A, CYP3A, and CYP2E1 were measured by ELISA; the mRNA expressions of TNF-α, IL-1ß, IL-6, and iNOS were determined by RT-PCR; and the relative protein expressions of c-Rel and p65 were analyzed by western blotting. Results showed that GLPS inhibited the elevations of the marker enzymes (GOT, GPT, LDH) and MDA induced by CCl4; it also enhanced the suppressed activity of antioxidant enzymes (SOD, CAT, GSH-Px, T-AOC). The treatment with GLPS resulted in significant downregulation of NF-κB and inflammatory cytokine mRNA levels and significant decreases in the hepatic protein levels of CYP1A, CYP3A, and CYP2E1. These results suggest that GLPS can protect CCl4-induced PCLS injury through inhibiting lipid peroxidation, elevating antioxidant enzyme activity, and suppressing immune inflammatory response.

Effects of carbon tetrachloride on oxidative stress, inflammatory response and hepatocyte apoptosis in common carp (Cyprinus carpio).

In the present study, the cellular and molecular mechanism of carbon tetrachloride (CCl4)-induced hepatotoxicity in fish was investigated by studying the effects of CCl4 on the oxidative stress, inflammatory response and hepatocyte apoptosis. Common carp were given an intraperitoneal injection of 30% CCl4 in arachis oil (0.5ml/kg body weight). At 72h post-injection, blood were collected to measure glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), liver samples were taken to analyze toll-like receptor 4 (TLR4), cytochrome P450 2E1 (CYP2E1) and gene expressions of inflammatory cytokines and nuclear factor-κB (NF-κB/cREL). Cell viability and apoptosis were analyzed after treatment of the primary hepatocytes with CCl4 at 8mM. The results showed that CCl4 significantly increased the levels of GPT, GOT, MDA, TLR4 and CYP2E1, reduced the levels of SOD, GPx, CAT, GSH and T-AOC, and up-regulated the gene expressions of NF-κB/cREL and inflammatory cytokines including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-1ß, IL-6 and IL-12. In vitro, CCl4 caused a dramatic loss in cell viability and induced hepatocyte apoptosis. Overall results suggest that oxidative stress lipid peroxidation, and TNF-α/NF-κB and TRL4/NF-κB signaling pathways play important roles in CCl4-induced hepatotoxicity in fish.

Grass carp reovirus induces apoptosis and oxidative stress in grass carp (Ctenopharyngodon idellus) kidney cell line.

Grass carp hemorrhage is an acute contagious disease caused by grass carp reovirus (GCRV). The pathogenesis of GCRV and the relationship between GCRV and the host cells remain unclear. The aim of the present study was to investigate the relations among apoptosis, intracellular oxidative stress and virus replication in GCRV infected-cells. The results showed that GCRV induced activation of caspase proteases as early as 12 h, and reached maximum activities at 24 h or 48 h post-infection in a grass carp kidney cell line (CIK cells). Meanwhile, the levels of tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß) also were increased in GCRV-infected CIK cells and showed a statistically significant difference from 24 h to 96 h post-infection. The infection of GCRV caused the destruction of entire monolayer and the death of host cells. Accompanied by the infection, a severe oxidative stress occurred, which led to extensive loss of antioxidants and formation of lipid peroxidation after 48 h post-infection. These data suggested that the apoptosis which was triggered at an early stage (12-24 h) in the viral infection cycle, might be independent of virus replication, while the oxidative stress induced by GCRV was mostly related to the virus replication.

The protective effect of silymarin on the carbon tetrachloride (CCl4)-induced liver injury in common carp (Cyprinus carpio).

Silymarin, a mixture of bioactive flavonolignans from the milk thistle (Silybum marianum), is traditionally used in herbal medicine to defend against various hepatotoxic agents. The aim of the present study was to evaluate the protective effect of silymarin against carbon tetrachloride (CCl4)-induced liver injury in fish. Common carp, with an average initial weight of 17.0 ± 1.1 g, were fed diet containing four doses of silymarin (0, 0.1, 0.5, and 1 g/kg diet) for 60 d. Fish were then given an intraperitoneal injection of CCl4 (30% in arachis oil) at a dose of 0.5 ml/kg body weight. At 72 h after CCl4 injection, blood and liver samples were collected for the analyses of serum biochemical parameters, liver index, peroxidation product, glutathione, and antioxidant enzyme activities. The results showed that administration of silymarin at 0.5 and 1 g/kg diet for 60 d prior to CCl4 intoxication significantly reduced the elevated activities of glutamate pyruvate transaminase, glutamate oxalate transaminase, lactate dehydrogenase (LDH), and increased the reduced levels of total protein and albumin in the serum. The reduced levels of liver index, superoxide dismutase, glutathione peroxidase, catalase, glutathione, and total antioxidant capacity were markedly increased, and malondialdehyde formation was significantly restrained in the liver. However, these parameters, except LDH, were not significantly changed in fish fed with silymarin at 0.1 g/kg diet. Based on the results, it can be concluded that silymarin has protective effect against CCl4-induced hepatotoxicity in fish. It is suggested that silymarin may be used as a hepatoprotective agent to prevent liver diseases in fish.

In vitro and in vivo hepatoprotective and antioxidant effects of Astragalus polysaccharides against carbon tetrachloride-induced hepatocyte damage in common carp (Cyprinus carpio).

The present study is aiming at evaluating the hepatoprotective and antioxidant effects of Astragalus polysaccharide (APS) on the carbon tetrachloride (CCl(4))-induced hepatocyte and liver injury in common carp in vitro and in vivo. In vitro, APS (200, 400 and 800 µg/ml) was added to the carp primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl(4) at 8 mM in the culture medium. APS at concentrations of 200, 400 and 800 µg/ml significantly improved cell viability and inhibited the elevation of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH) and malondialdehyde (MDA) and significantly increased the reduced level of superoxide dismutase (SOD). In vivo administration of APS at the doses of 1.5 and 3 g/kg in the diet for 60 days prior to CCl(4) intoxication significantly reduced the elevated activities of GPT, GOT and LDH and increased the reduced levels of total protein and albumin in the serum; meanwhile, the reduced levels of SOD, glutathione and total antioxidant capacity (T-AOC) were markedly increased and the MDA formation was significantly inhibited in liver tissue. Overall results proved the hepatoprotective action of APS, which is likely related to its antioxidant activity. The results support the use of APS as a hepatoprotective and antioxidant agent in fish.

Hepatoprotective and antioxidant effects of Glycyrrhiza glabra extract against carbon tetrachloride (CCl(4))-induced hepatocyte damage in common carp (Cyprinus carpio).

The present study is aiming at evaluating the hepatoprotective and antioxidant effects of Glycyrrhiza glabra extract (2.5, 5 and 10 µg/ml) on the carbon tetrachloride (CCl(4))-induced carp hepatocyte damage in vitro. Glycyrrhiza glabra extract was added to the carp primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl(4). CCl(4) at 8 mM in the culture medium produced significantly elevated levels of lactate dehydrogenase (LDH), glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT) and malondialdehyde (MDA) and significantly reduced levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Pre-treatment (5 µg/ml) and pre- and post-treatment (5 and 10 µg/ml) of the hepatocytes with Glycyrrhiza glabra extract significantly reduced the elevated levels of LDH, GOT, GPT and MDA and increased the reduced levels of SOD and GSH-Px by CCl(4); post-treatment of the hepatocytes with Glycyrrhiza glabra extract at 5 µg/ml reduced the GPT and GOT levels and increased the GSH-Px level, but had no effect on the other parameters at all the studied concentrations. The results support the use of Glycyrrhiza glabra extract as a hepatoprotective and antioxidant agent in fish.

Hepatoprotective and antioxidant effects of Hibiscus sabdariffa extract against carbon tetrachloride-induced hepatocyte damage in Cyprinus carpio.

The present study aims to evaluate the hepatoprotective and antioxidant effects of Hibiscus sabdariffa extract on the carbon tetrachloride (CCl(4))-induced hepatocyte damage in fish and provide evidence as to whether it can be potentially used as a medicine for liver diseases in aquaculture. H. sabdariffa extract (100, 200, and 400 µg/mL) was added to the carp primary hepatocyte culture before (pre-treatment), after (post-treatment), and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl(4). CCl(4) at 8 mM in the culture medium produced significantly elevated levels of lactate dehydrogenase (LDH), glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), and malondialdehyde (MDA) and significantly reduced levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Pre-treatment and pre- and post-treatment of the hepatocytes with H. sabdariffa extract significantly reduced the elevated levels of LDH, GOT, GPT, and MDA and increased the reduced activities of SOD and GSH-Px in a dose-dependent manner; post-treatment did not show any protective effect. The results suggest that H. sabdariffa extract can be potentially used for preventing rather than curing liver diseases in fish.

Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish.

A recombinant protein for the S-layer protein of Aeromonas hydrophila was produced and its ability to protect common carp Cyprinus carpio L. against six virulent isolates of A. hydrophila was assessed. A group of 120 carp (30-40 g) were vaccinated intra-peritoneally with 0.1 ml of adjuvanted vaccine (30 microg protein per fish). Another group of 120 carp were injected with 0.1 ml of PBS-adjuvant mixture to serve as controls. Twenty fish from each group were challenged with each one of six virulent isolates of A. hydrophila 35 days post-vaccination. The fish were maintained in 12 separate tanks before terminating the experiment at 16 days post-challenge. The relative percentage survival (RPS) for the six isolates of A. hydrophila ranged from 56 to 87%. The difference in survival rate of fish challenged with four of the isolates was statistically significant in vaccinated fish compared to control fish, when analysed using a Chi-square test. The results of the study suggest that the recombinant S-layer protein of A. hydrophila could be useful as a vaccine antigen to protect fish against different isolates of this pathogenic bacterium.

Immune responses of resistant and sensitive common carp families following experimental challenge with Aeromonas hydrophila.

Parameters of non-specific immune response and level of specific and natural antibodies were investigated after an experimental challenge with Aeromonas hydrophila in genetically different common carp (Cyprinus carpio) families. Ten resistant and ten sensitive families were used for the experiment, which had been selected out of 96 families, based on the results of a preliminary challenge test. Blood samples were collected 12 h, one week and 21 days following the challenge. Phagocytic and respiratory burst activities of phagocytic cells, lysozyme activity of the blood plasma were determined. Level of specific antibodies against A. hydrophila and level of natural antibodies were measured in the samples taken on the 28th day. Non-infected fish from resistant and sensitive families were used as controls. Significant differences of phagocytic and lysozyme activities were measured between the challenged resistant and sensitive families. The level of specific antibodies between the same families was also found to be significantly different. There were no significant differences of the studied parameters between the control groups. Based on our results, phagocytic activity of leukocytes, plasma lysozyme activity and specific antibody titre were found to be higher in the resistant families than in the sensitive ones following infection with A. hydrophila.

Differences in the antibody response and survival of genetically different varieties of common carp (Cyprinus carpio L.) vaccinated with a commercial Aeromonas salmonicida/A. hydrophila vaccine and challenged with A. hydrophila.

Males of two strains of carp, wild Duna (D), and inbred Szarvas 22 (22), were selected for high and low stress response. Two purebreds of D and 22, from randomly chosen parents and four crosses, 22 x 22-L (low stress response), 22 x 22-H (high stress response), 22 x D-L (low stress response) and 22 x D-H (high stress response) from selected stress response parents were produced and vaccinated with a commercial Aeromonas salmonicida/Aeromonas hydrophila vaccine and their circulating antibody response evaluated 1, 3, 5, and 7 weeks post-vaccination by ELISA. Significantly higher titres of circulatory antibodies against A. hydrophila were found in the families 22 and cross 22 x 22-L compared to other groups. The development of circulatory antibodies against A. hydrophila in all crosses having at least one D parent was low and remained low throughout the experiment. The level of circulatory antibodies against atypical A. salmonicida in the inbred strain increased following a booster vaccination with the highest values measured in inbred strain 22 and cross 22 x 22 L. The different varieties of carp had different levels of survival against experimental challenge with A. hydrophila. The greatest survival was obtained in strain 22 and cross 22 x 22-L, while ~90% of D wild carp and cross 22 x D (independent of their stress response) died. Survival results correlated well with the antibody response of the different groups: 22 and 22 x 22-L had the highest antibody titres against A. hydrophila and the greatest level of survival.

Protein expression by Aeromonas hydrophila during growth in vitro and in vivo.

Expression of Aeromonas hydrophila cellular and extracellular products (ECPs) was examined following culture of the bacterium in vitro, in Tryptic Soy Broth (TSB), and in vivo, in dialysis tubing placed within the peritoneal cavity of common carp (Cyprinus carpio L.). Whole cell (WC), outer membrane proteins (OMPs) and ECP components of the bacteria were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations. The aim of the study was to identify unique and common proteins up-regulated in vivo. Unique bands were seen in the 1D gels at 58 and 55 kDa for WC and OMP preparations, respectively, for all the four virulent and two avirulent isolates cultured in vivo. Bands of increased intensity were also observed at 70, 55, 50 and 25 kDa with WC preparations for all virulent isolates cultured in vivo. Analysis of WC by 2D SDS-PAGE revealed that bacteria cultured in vivo expressed a number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0 to 6.0. The unique proteins identified in vivo may be involved in the virulence of the bacterium and their potential as vaccine candidates is currently being investigated.

Effect of pulp and paper mill effluent (BKME) on physiological parameters of roach (Rutilus rutilus) infected by the digenean Rhipidocotyle fennica.

Physiological parameters were measured after experimental infection of roach (Rutilus rutilus L.) with Rhipidocotyle fennica Gibson, Valtonen et Taskinen, 1992 (Digenea) cercariae. The fish were caught from two lakes: a eutrophic bleached kraft mill effluent (BKME)-contaminated lake and an oligotrophic unpolluted lake. The intensity of infection was followed up to 10 days post infection (p.i.) and physiological parameters indicating non-specific stress responses and the condition of fish were examined simultaneously. The mean abundance, the number of parasites per fish, of R. fennica was significantly higher in the fish from the contaminated water during the first two days p.i., probably reflecting the decreased resistance of these fish to infection. The decrease of leukocrit, as well as the increase of the activity of transaminases (GOT and GPT) in infected fish of both groups are suggestive of pathological processes caused by cercariae penetrating the fish. A significantly lower leukocrit value, as well as higher alkaline phosphatase activity and plasma chloride levels were noted in fish originating from the contaminated lake compared to those from the unpolluted lake. No significant differences were noted in haematocrit, plasma protein and calcium values between the fish from the uncontaminated and contaminated lakes, or between the infected and uninfected control fish.