The Essential Oil of Hyptis mutabilis in Ichthyophthirius multifiliis Infection and its Effect on Hematological, Biochemical, and Immunological Parameters in Silver Catfish, Rhamdia quelen.

This study evaluated the activity of leaf essential oil (EO) from Hyptis mutabilis as well as its major constituent, (-)-globulol, in infections by the parasite Ichthyophthirius multifiliis (ich). Effects on hematological, biochemical, and immunological parameters of silver catfish, Rhamdia quelen, exposed to the same samples also were evaluated. In the first experiment, naturally infected fish were treated with EO (0, 10, and 20 mg · L-1) and ethanol, using several methods of exposure. Fish mortality and the number of trophonts per fish were assessed after 48 and 96 hr. Hour-long daily baths resulted in optimal survival, so this methodology was used for the second experiment, in which infected animals were exposed to (-)-globulol at 2.5 and 5 mg · L-1. The most effective concentrations in Experiments 1 and 2 were chosen for Experiment 3, in which healthy animals were subjected to hour-long daily baths with EO (20 mg · L-1) or (-)-globulol (2.5 mg · L-1). Additionally, an in vitro experiment was performed with EO and globulol at the same concentrations of the in vivo test. EO and (-)-globulol increased the survival of fish infected with ich and altered certain hematological and biochemical parameters. After 4 days, levels of hematocrit, erythrocytes, and leukocytes increased significantly in healthy animals exposed to EO. Exposure to (-)-globulol increased leukocyte number alone. No significant differences in nonspecific immunological parameters were detected when treated groups were compared to controls, but the leukocytosis observed in EO- and globulol-treated healthy animals indicates that EO and (-)-globulol increased innate immunity in these fish. An in vitro antiparasitic effect was observed in both samples.

Effect of dietary supplementation with Suaeda maritima on blood physiology, innate immune response, and disease resistance in olive flounder against Miamiensis avidus.

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder.

Inonotus obliquus containing diet enhances the innate immune mechanism and disease resistance in olive flounder Paralichythys olivaceus against Uronema marinum.

The present study describes the effect of diet supplementation with Chaga mushroom, Inonotus obliquus extract at 0%, 0.01%, 0.1%, and 1.0% levels on the innate humoral (lysozyme, antiprotease, and complement), cellular responses (production of reactive oxygen and nitrogen species and myeloperoxidase), and disease resistance in olive flounder, Paralichythys olivaceus against Uronema marinum. The lysozyme activity and complement activity significantly increased in each diet on weeks 2 and 4 against pathogen. The serum antiprotease activity and reactive nitrogen intermediates production significantly increased in fish fed with 0.1% and 1.0% diets from weeks 1-4. However, reactive oxygen species production and myeloperoxidase activity significantly increased in 1.0% and 2.0% diets on weeks 2 and 4. In fish fed with 0.1% and 1.0% diets and challenged with U. marinum the cumulative mortality was 50% and 40% while in 0% and 0.01% diets the mortality was 85% and 55%. The results clearly indicate that supplementation diet with I. obliquus at 0.1% and 1.0% level positively enhance the immune system and confer disease resistance which may be potentially used as an immunoprophylactic in finfish culture.

Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum).

The effects on rainbow trout, Oncorhynchus mykiss (Walbaum), immune parameters by differently formulated fish feed types containing immunostimulants have been tested in a double-blind, duplicated and controlled study performed over 50 days. A total of 800 rainbow trout (10-12 g) were kept in eight duplicate fish tanks (each containing 100 fish) and fed at a daily feeding rate of 1.5% of the biomass. The feed types were (1) control feed (C) without additives, (2) feed containing beta-glucan, nucleotides, manno-oligosaccharides (MOS), vitamins C and E (GNMCE), (3) feed containing probiotic bacteria and plant extracts (PP) and (4) feed with nucleotides, manno-oligosaccharides, vitamins C and E (NMCE). Plasma lysozyme activity was increased in fish fed two feed types (GNMCE and NMCE) but slightly depressed in fish fed PP. A non-significant trend for a higher mucous cell density at days 30 and 50 was shown in all fish receiving feeds with additives compared to the control group. All fish became infected with Ichthyophthirius multifiliis when exposed, but fish fed GNMCE showed a significantly lower infection both at days 30 and 50. Expression of genes encoding C3 and MHCII was significantly up-regulated in fish fed GNMCE for 50 days, and the expression of genes coding Hepcidin was significantly down-regulated in fish fed NMCE for 50 days. Beta-glucan was the single component, when used in combination with other feed ingredients, which was found associated with increased parasite resistance, increased lysozyme and immune gene up-regulation.

In vivo innate immune responses of groper (Polyprion oxygeneios) against Miamiensis avidus infection and lack of protection following dietary vitamin C administration.

Scuticociliates are extracellular histophagous parasites that affect farmed fish worldwide. One of the most common pathogenic species is Miamiensis avidus, a pathogen of New Zealand groper (Polyprion oxygeneios). The aim of this study was to characterise both the host (groper)-parasite (M. avidus) immune interactions and the possible protective role of dietary sodium ascorbate. Head-kidney leucocytes (HKLs) from naturally infected adult groper showed decreased respiratory burst response and peroxidase (Px) levels than healthy individuals. Infected groper also had significantly higher serum Px levels compared to controls. Myeloperoxidase (MPO) was inhibited in the head-kidney (HK) whereas MPO(+) cells were observed in the skin and muscle lesions. The inhibition of the innate immune responses was further studied in experimental infections with M. avidus, which confirmed depletion of Px inside leucocytes and marked increases in serum Px in infected individuals. Groper juveniles were fed a diet supplemented with sodium ascorbate (Vitamin C) (2g Kg(-1)) for 21 days and then challenged by subcutaneous injection or immersion exposure with live M. avidus cells. No protection was observed in the sodium ascorbate fed groper compared to the control diet following challenge by either injection or immersion. In vitro assays showed that sodium ascorbate itself results in the inhibition of Px and respiratory burst of groper HKLs, supporting the results obtained in vivo. Our results show that histophagous protozoa such as M. avidus hamper innate immune defences of fish hosts and that dietary sodium ascorbate does not protect groper against experimental infection with this parasite.

Effects of dietary n-3 highly unsaturated fatty acids on growth, nonspecific immunity, expression of some immune related genes and disease resistance of large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans).

The study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acid (n-3 HUFA) on growth, nonspecific immunity, expression of some immune related genes and disease resistance of juvenile large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans). Six isoproteic and isolipidic diets were formulated with graded levels of n-3 HUFA ranging from 0.15% to 2.25% of the dry weight and the DHA/EPA was approximately fixed at 2.0. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.0 × 1.0 × 1.5 m), and each cage was stocked with 60 fish (initial average weight 9.79 ± 0.6 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 58 days. Results showed that moderate n-3 HUFA level (0.98%) significantly enhanced growth compared with the control group (0.15% HUFA) (P < 0.05), while higher n-3 HUFA levels (1.37%, 1.79% and 2.25%) had detrimental effects on the growth though no significance was found (P > 0.05). Nitro blue tetrazolium (NBT) positive leucocytes percentage of head kidney and serum superoxide dismutase (SOD) activity increased with increasing n-3 HUFA from 0.15% to 0.60%, and decreased with further increase of n-3 HUFA from 0.60% to 2.25% (P < 0.05). Serum lysozyme activity increased significantly as n-3 HUFA increased from 0.15% to 1.37%, and then decreased with n-3 HUFA from 1.37% to 2.25% (P > 0.05). There were no significant differences in phagocytosis index (PI) of head kidney leucocytes among dietary treatments (P > 0.05). The hepatic mRNA expression of Toll-like receptor 22 (TLR22) and Myeloid differentiation factor 88 (MyD88) was significantly up-regulated in fish fed the diets with low or moderate levels, while in kidney this increment was only found at specific sampling time during the natural infestation of parasites. The 13 d cumulative mortality rate following natural infestation of parasites decreased with n-3 HUFA increased from 0.15% to 0.60% (P < 0.05), and significantly increased with n-3 HUFA from 0.60% to 2.25% (P < 0.05). Results of this study suggested that fish fed low or moderate dietary n-3 HUFA had higher growth, nonspecific immune responses, expression levels of some immune related genes and disease resistance of large yellow croaker following natural infestation of parasites and dietary n-3 HUFA may regulate fish immunity and disease resistance by altering the mRNA expression levels of TLR22 and MyD88.

Styrax japonica supplementation diet enhances the innate immune response in Epinephelus bruneus against bacterial and protozoan infections.

Kelp grouper, Epinephelus bruneus, fed for 30 days with 0% (control), 0.1%, 1.0%, and 2.0% of Styrax japonica supplementation diets, led to reductions in mortality after being challenged with a bacterium (Vibrio harveyi) and a ciliate protozoan (Uronema marinum). The enriched diets significantly increased the survival rate as compared to the controls. The phagocytic and respiratory activities were significantly increased in kelp groupers given 1.0% and 2.0% enriched diets. The complement activity, lysozyme activity, serum bactericidal activity, and total protein level significantly increased with any enriched diet against the pathogens; however antiprotease activity and myeloperoxidase levels significantly increased only with 1.0% and 2.0% enriched diets while the α2-macroglobulin level was significantly enhanced with 1.0% enriched diet. The study suggests that incorporation of S. japonica at 1.0% and 2.0% level in the diet significantly enhances the immune responses in the kelp grouper E. bruneus against V. harveyi and U. marinum.

Korean mistletoe enriched diet enhances innate immune response in kelp grouper, Epinephelus bruneus against Philasterides dicentrarchi.

The present study investigated the immunostimulatory effect of Korean mistletoe Viscum album extract (KM-E) on innate immune response in kelp grouper Epinephelus bruneus against Philasterides dicentrarchi. Kelp grouper were divided into four groups of 25 each and fed with 0 (control), 0.5, 1.0, and 2.0% enriched diets with Korean mistletoe extract (KM-E). After feeding for 30 days, the fish were injected intraperitoneally (i.p.) with 100 µl of P. dicentrarchi (4.2 × 10(7)ciliates/ml) to study the immune responses at weeks 1, 2, and 4. The respiratory burst activity did not significantly enhance when fed with 0.5% and 1.0% supplementation diets on week 1 when compared to control diet. On weeks 2 and 4, the respiratory burst activity significantly increased with 1.0% and 2.0% diets. The phagocytic activity significantly enhanced with 1.0% and 2.0% diets, but not with 0.5% diet at any time. When fed with 1.0% KM-E-diet the lysozyme activity did not significantly vary at any week whereas with 1.0% and 2.0% diets it was significantly enhanced. The total protein level significantly increased with 1.0% and 2.0% KM-E-diets from weeks 1 to 4 as compared to control. The present study suggests that 1.0% or 2.0% KM-E-supplementation diet positively enhances the innate immune response in E. bruneus against P. dicentrarchi infection.

Kalopanax pictus as feed additive controls bacterial and parasitic infections in kelp grouper, Epinephelus bruneus.

Feeding kelp grouper, Epinephelus bruneus (26.1 ± 1.4), with 0%, 0.1%, 1.0%, and 2.0% Kalopanax pictus extract-supplementation diets, for 30 days reduced mortality on being challenged intraperitoneally 100 µl with bacterium Vibrio alginolyticus (2.1 × 10(7) cfu ml(-1)) and ciliate parasite Philasterides dicentrarchi (2.3 × 10(7) ciliates ml(-1)). The red blood cells (RBC), white blood cells (WBC), haemoglobin, haematocrit, lymphocytes, and monocytes levels significantly increased in kelp grouper fed with all doses of K. pictus-supplementation diets and challenged with bacterium and parasite when compared to control. However, the levels of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), neutrophils, and thrombocytes did not significantly. The phagocytic activity, complement activity, and antiprotease activity did not significantly change in kelp grouper fed with 0.1% K. pictus-supplementation diets and challenged with bacterium and parasite. The respiratory activity, lysozyme activity, bactericidal activity, total protein level, and myeloperoxidase levels significantly increased in kelp grouper fed with all the doses of K. pictus-supplementation diet and challenged with bacterium and parasite. However, α2-macroglobulin level significantly increased with 1.0% diet, but not with 0.1% and 2.0% diets. Therefore this study suggests that 1.0% and 2.0% K. pictus-supplementation diets positively protected and enhanced the immune system in kelp grouper E. bruneus against V. alginolyticus and P. dicentrarchi infection.

Effect of traditional Korean medicinal (TKM) triherbal extract on the innate immune system and disease resistance in Paralichthys olivaceus against Uronema marinum.

We report the effect of aqueous-, ethanol- and methanol-solvent-derived extracts of three traditional Korean herbs, Punica granatum, Chrysanthemum cinerariaefolium and Zanthoxylum schinifolium, by monitoring the innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and the functional immunity in terms of percentage mortality and relative percent survival (RPS) in olive flounder (Paralichthys olivaceus) against Uronema marinum (1 x 10(5)ciliates ml(-1)) for 30 days. Fish were intraperitoneally administered with 5, 50 and 100 mg kg(-1) body weight of each traditional Korean medicinal (TKM) solvent extract except the control and infected untreated groups. In all the treated groups at concentrations of 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters were found significantly enhanced when compared to 0 mg kg(-1) dose. However, at 5 mg kg(-1) the tested immune parameters did not vary. Administration of TKM solvent extracts preceding the challenge with U. marinum for 30 days significantly reduced the percentage mortality with the consequent increase in RPS. Administration of 50 and 100 mg kg(-1) TKM solvent extracts clearly enhanced the innate immune responses and disease resistance in P. olivaceus against U. marinum.

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection.

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.