Galina Jeney

Prevention of stress in rainbow trout (Oncorhynchus mykiss) fed diets containing different doses of glucan

An experiment was performed to determine the effects of stress on non-specific defence mechanisms in rainbow trout fed diets containing different doses of glucan. Fish were fed with 0, 0.1, 0.5 and 1.0% glucan concentration in food. After 4 weeks of feeding, fish were stressed by 2 h transportation. The effect of stress was investigated by measuring changes in blood levels of cortisol, glucose, total protein and composition of their leukocyte population, as well as by changes in non-specific defence mechanisms of the fish. After 4 weeks feeding with glucan, elevated phagocytosis and oxidative radical production were observed in treated fish, but the levels did not correlate with the different doses of glucan. Stress induced by 2 h transportation caused high cortisol levels in plasma and hyperglycaemia in all groups, but the lowest level of glucose was measured in the group fed the low (0.1%) dose of glucan. Respiratory burst activity, phagocytosis, serum protein and lyzosyme levels were found to be significantly reduced by stress. The most dramatic reduction was observed in the control group, but the changes were not affected by glucan doses. One week post stress, hyperglycaemia was still observed in control and fish fed medium and high doses of glucan. Further reductions of total protein and intracellular oxidative radical production, were measured in all groups, but in fish fed with low dose of glucan the changes were less dramatic. The phagocytosis ratio increased in all groups, but did not attain the levels measured before stress in control and in the group fed the high concentration of glucan. A spontaneous infection with Flexibacter columnaris caused mortality in all groups except the group fed the low level of glucan. The results of the present study show that feeding of glucan in low doses several weeks before transportation can help to prevent negative effects of stress.

Glucan injection or bath exposure given alone or in combination with a bacterin enhance the non-specific defence mechanisms in rainbow trout (Oncorhynchus mykiss)

Abstract Elevations in the non-specific defence mechanisms were noted after trout were injected or bathed in glucan solutions or in solutions containing the glucan combined with a bacterin, Y. ruckeri O-antigen. Periodic non-lethal blood samples showed that neutrophil activity as evaluated by the ability of the cells to stick to glass and produce oxidative radicals as detected by the nitroblue tetrazolium (NBT) assay, rose after treatments within 2 days. Elevated phagocytosis, assessed by increased uptake of glutaraldehyde-treated sheep red blood cells, also confirmed these kinetics. The numbers of circulatory glass-adherent cells from fish given the glucans by injection or bath was twice the level of the sham-treated controls; likewise the phagocytic ratio also was two-fold higher. In following the kinetics of the non-specific defensive responses, the injection of the glucan caused an immediate, slight reduction of NBT staining cells and numbers of leukocytes before the 2–3 day rise; this was not obvious in the fish given the glucan by bath. The glucans could play an important role in the prevention of diseases in fish culture.

Recent achievements in studies on diseases of common carp (Cyprinus carpio L.)

Abstract Parasitic, fungal, bacterial and viral diseases of common carp (Cyprinus carpio L.) are reviewed. Besides a general overview of parasites of carp, swimbladder inflammation, caused by Sphaerospora spp., is discussed in detail. Saprolegnia spp. is the most important fungal pathogen. Aeromonas hydrophila and the atypical Aeromonas salmonicida, as well as Flexibacter columnaris, are described as the major bacterial pathogens of carp. Spring viraemia of carp caused by Rhabdovirus carpio is presented as the main viral infection of common carp. Details on methods of treatment and prevention are presented together with a description of the given diseases. The role of environmental stress, including “normal” culture practice and pollution, in the outbreak of diseases of common carp is discussed. Prospective methods to minimize the risk of diseases as well as their limitations are presented.

Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection in rainbow trout (Oncorhynchus mykiss).

Immunostimulants were given to rainbow trout for assaying effects on modulating non-specific defense mechanisms, specific immune response, and protection levels against pathogen challenge with Aeromonas salmonicida. Three drugs, levamisole (an approved veterinary drug in the USA), a quaternary ammonium compound (QAC), and a short-chain polypeptide (ISK) were found to affect the non-specific defense mechanism activities, which were measured by changes in circulatory neutrophil and phagocytic activity levels, and the specific immune response factors, which were measured by numbers of plaque-forming cells, and circulatory antibody levels. When given alone, the immunostimulants elevated the non-specific factors. When injected in combination with an A. salmonicida O-antigen bacterin, the non-specific factors were further elevated, and the specific response was raised over samples taken from fish given the bacterin without the immunostimulants. Challenge tests with the virulent pathogen, A. salmonicida, showed a 5-6 day delay in the onset of mortalities in the fish given the immunostimulants alone, and a 12-14 day delay when immunostimulants given were combined with the bacterin. In the groups given the QAC or ISK with the bacterin, there was a 20% and 40% survival rate, respectively.

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.

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-1β, 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.

An in vitro technique for surveying immunostimulants in fish

Abstract In vitro assays were developed to investigate the effects of immunostimulants on leukocytes in spleen sections of rainbow trout ( Oncorhynchus mykiss ). Fish spleens were divided, and individual sections were placed in 10 ml of tissue culture media and 0.1, 1, 10, or 100 μg/ml dilutions of Levamisole, QAC (quaternary ammonium compound), or ISK (a polypeptide). After 4 days of incubation, cell suspensions were prepared to examine the effects of the immunostimulants on neutrophil oxidative activity using the nitroblue tetrazolium (NBT) assay and on phagocytic activity by engulfment of glutaraldehyde-fixed sheep red blood cells. QAC induced a heightened activity in plates receiving 0.1 and 1.0 μg/ml, but suppressed activity at 10 and 100 μg/ml levels. Sections incubated with ISK showed heightened responses at all doses except 100 μg/ml. Levamisole showed heightened responses at all doses in the NBT assay, and at the 1.0 and 10 μg/ml doses in the phagocytic assay.

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.

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-1β (IL-1β) 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.

Adjuvants and Immunostimulants for Potentiating Protection Against Furunculosis in Fish

Publisher Summary Effective bacterins against furunculosis disease in salmonids are now commercially available. The adjuvants are the substances having potentiating effects on the specific immune response and are used with vaccines. When these adjuvants are used without vaccine, these are termed as “immunostimulants.” The addition of adjuvant heightens the humoral antibody titres and enhances protection against furunculosis, that is, increases the effectiveness of bacterins used against furunculosis. Complete Freunds adjuvant (CFA) was used as the important potentiating component for the first time. Beta-glucans, muramyl dipeptide, streptococcal extracts, light-oils, and levamisole induce marked elevations of the non-specific defence mechanisms, as well as the specific immune response of fish. The immunostimulants affect the nonspecific defense mechanisms. The CFA when injected without a bacterin has the capability to induce significant protection against furunculosis. Nonspecific defense mechanisms, including phagocytic cell adherence, oxidative radical release, and engulfment, are among the most important early cellular factors in protection.