Pharmacokinetic behaviour and pharmacokinetic-pharmacodynamic integration of doxycycline in rainbow trout (Oncorhynchus mykiss) after intravascular, intramuscular and oral administrations.

OBJECTIVE: Doxycycline (DO) has been used in fish for a long time, but there are some factors that have not yet been clarified regarding its pharmacokinetic (PK) and pharmacodynamic (PD) properties. Therefore, the aim of this study was to investigate the PK and PK/PD targets of DO after 20 mg/kg intravascular (IV), intramuscular (IM) and oral (OR) gavage administration in rainbow trout (Oncorhynchus mykiss). METHODS: Plasma samples were collected at specific time points and subsequently analysed by HPLC-ultraviolet. The PK/PD indices were calculated based on the MIC90 (Aeromonas hydrophila and Aeromonas sobria) values obtained for the respective bacteria and the PK parameters obtained for DO following both IM and OR administration. RESULTS: After IV administration, the elimination half-life (t1/2 ʎz), area under the concentration vs. time curve (AUC), apparent volume of distribution at steady-state and total body clearance of DO were 34.81 h, 723.82 h µg/mL, 1.24 L/kg and 0.03 L/kg/h, respectively. The t1/2λz of the DO was found to be 37.39 and 39.78 h after IM, and OR administration, respectively. The bioavailability was calculated 57.02% and 32.29%, respectively, after IM and OR administration. The MIC90 of DO against A. hydrophila and A. sobria was 4 µg/mL. The PK/PD integration showed that DO (20 mg/kg dose) for A. hydrophila and A. sobria with MIC90 ≤4 µg/mL achieved target AUC/MIC value after IM administration. CONCLUSIONS: These results suggest that when rainbow trout was treated with 20 mg/kg IV and IM administered DO, therapeutically effective concentrations were reached in the control of infections caused by A. hydrophila and A. sobria.

Dietary flaxseed (Linum usitatissimum) oil supplementation affects growth, oxidative stress, immune response, and diseases resistance in rainbow trout (Oncorhynchusmykiss).

This paper describes the effects of flaxseed (Linum usitatissimum) oil (FSO) as a feed additive on growth performance, oxidative stress, immunity, and disease resistance in rainbow trout (Oncorhynchus mykiss). Eight-hundred-and-forty rainbow trout individuals (mean weight: 25.66 ± 1.33 g) were fed with different doses of FSO (0.5, 1, and 1.5%) ad libitum two times a day for 9 weeks. At the end of the feeding, growth performance was evaluated and the fish were challenged with two different bacteria (Yersinia ruckeri and Aeromonas hydrophila). At the end of the 3rd, 6th, and 9th weeks, blood and tissue samples were taken from 9 fish per treatment to evaluate innate immune response, cytokine gene expression levels, antioxidant enzyme activities and lipid peroxidation levels, and digestive enzyme activities. Determination of haematological parameters and histological examination was also carried out to evaluate the general health status of the fish. Results showed that the final weight and specific growth rate of FSO-supplemented fish increased significantly (p < 0.05). FSO-supplemented fish showed higher resistance to Y. ruckeri than the control group (p < 0.05). However, survival rates of all groups in A. hydrophila challenge test were similar (p > 0.05). Among the investigated innate immune response parameters, the potential killing activity of phagocytes, myeloperoxidase activity, and lysozyme activity increased in the FSO-supplemented groups (p < 0.05). Almost all cytokine gene expression levels in the experimental groups up-regulated especially after 9 weeks of feeding in the head kidney and intestine (p < 0.05). Similarly, superoxide dismutase and catalase activities were found to be significantly higher in the FSO group than in the control (p < 0.05) whereas, the lipid peroxidation levels drastically declined as a result of the FSO supplementation (p < 0.05). These results suggest that FSO can improve growth, enhance immune response, and lower oxidative damage in rainbow trout when supplemented at the rates of 0.5-1.5% for 9 weeks.

Effect of dietary celery (Apium graveolens) on the growth performance, immune responses, and bacterial resistance against Vibrio anguillarum of European seabass (Dicentrarchus labrax).

In this study, we evaluated to reveal the effects of aqueous methanolic extract of celery (Apium graveolens) on the growth performance, immune responses, and resistance against Vibrio anguillarum in European seabass (Dicentrarchus labrax). For this purpose, twenty fish (initial mean weight of 4.80 ± 0.06 g) were placed into twelve tanks (400 L) in triplicate and fish were fed with control (C) and three different levels (0.01, 0.05, and 0.1 g/kg) of A. graveolens (AG) extract-containing diets (AG0.01, AG0.05, and AG0.1) for 30 days. Blood and tissue (kidney, spleen, and intestine) samples were taken from the fish every 10 days during the study to determine the immune responses of the fish. Respiratory burst activity (RBA) was significantly decreased in the AG0.1 group compared to all other groups on the 10th day of the study (P < 0.05). Significance was noticed in the RBA of fish in all AG groups compared to the C group (P < 0.05) on the 30th day of the experiment Lysozyme activity (LYS) was raised on the 10th day of the study in all celery groups compared to the C group (P < 0.05). No differences in the myeloperoxidase activity (MPO) were observed among the experimental groups (P > 0.05). The final mean weight (FMW) was not affected in any experimental groups (P > 0.05). However, in the AG0.05 group, the specific growth rate (SGR) increased, and the feed conversion ratio (FCR) decreased compared to other groups (P < 0.05). IL-1ß in the kidney was highly elevated in the AG0.01 group on the 20th day of the study (P < 0.05). Similar results were observed on IL-6, IL-8, and TNF-α expression in the kidney (P < 0.05). Anti-inflammatory responses (IL-10 and TGF-ß) also increased in all experimental groups and tissues compared to the C group (P < 0.05). COX-2 was upregulated on the 20th day of the study in all tissues (P < 0.05). At the end of the feeding trial, the survival rate of the AG0.1 group in fish infected with Vibrio anguillarum infection was higher than the C group. Dietary celery extract did not affect growth performance directly but increased innate immune responses and a high survival rate. Overall, compared to the control group, the growth, immunity, and resistance of European seabass fed with a diet containing 0.05 g/kg celery aqueous methanolic extract has been improved, and this could be used as an immunostimulant feed additive.

Effects of white mustard (Sinapis alba) oil on growth performance, immune response, blood parameters, digestive and antioxidant enzyme activities in rainbow trout (Oncorhynchusmykiss).

A study was conducted to evaluate the effects of dietary supplementation of white mustard (Sinapis alba) oil (WMO) on growth performance, immune responses, digestive and antioxidant enzyme activities in juvenile rainbow trout (Oncorhynchus mykiss). For this purpose, fish (initial weight: 25.77 ± 0.13 g) were divided into four experimental groups in triplicate and fed ad libitum twice a day with diets containing WMO at 0 (control), 0.5, 1, and 1.5% of diet for 9 weeks. Three fish from each tank (n:9 per treatment) were sampled on 21st, 42nd, and 63rd days for further analyses. At the end of the feeding period, fish were challenged with Aeromonas hydrophila and Yersinia ruckeri in two separate experimental setups. Results showed that final weight, weight gain, and specific growth rate were significantly increased in all experimental groups compared to the control. Feed conversion ratio was similar among treatments. Respiratory burst and potential killing activity decreased in all experimental groups compared to the control (P < 0.05). Lysozyme and myeloperoxidase activities were elevated in all experimental groups at the end of the experiment compared to the control (P < 0.05). Cytokine gene expressions in the head kidney and intestine were elevated in all experimental groups compared to that of the control in general (P < 0.05). Hematological responses of the experimental fish groups were similar to that of the control (P > 0.05). Pepsin and trypsin levels decreased in all experimental groups (P < 0.05). In terms of antioxidant enzyme activities, significant improvement in liver superoxide dismutase, catalase, and glutathione s-transferase activities in all treatment groups were determined (P < 0.05). In addition, a significant decline in liver lipid peroxidation levels was recorded in all treated groups at all sampling times compared to the control (P < 0.05). At the end of this feeding trial, no significant differences (P > 0.05) were observed in survival against A. hydrophila among experimental groups compared to the control (P > 0.05). However, increased survival against Y. ruckeri was determined in experimental fish groups (P < 0.05). This study suggests that white mustard oil had a favorable effect on the overall health and growth of rainbow trout.

Plasma and muscle tissue disposition of enrofloxacin in Nile tilapia (Oreochromis niloticus) after intravascular, intraperitoneal, and oral administrations.

The aim of the study was to investigate the plasma and muscle pharmacokinetic of enrofloxacin (ENR) and its active metabolite ciprofloxacin (CIP) in Nile tilapia (Oreochromis niloticus) following single intravascular (IV), intraperitoneal (IP), or oral (PO) administration at 30 ± 1 °C. In this study, 234 healthy Nile tilapia (120-150 g) were used. The fish received a single IV, IP, or PO treatment of ENR at a dose of 10 mg/kg. The plasma and muscle tissue concentrations of ENR and CIP were measured using high-performance liquid chromatography with fluorescence detection and were evaluated using non-compartmental analysis. The elimination half-life, volume of distribution at steady state, and total body clearance of ENR were 21.7 h, 2.69 L/kg, and 0.09 L/h/kg, respectively. The peak plasma concentrations of ENR after IP or PO administration were 6.11 and 4.21 µg/mL at 0.25 and 2 h, respectively. The bioavailability of ENR for IP or PO routes was 78% and 86%, respectively. AUC(0-120)muscle/AUC(0-120)plasma ratios following the IV, IP, or PO administrations were 1.43, 1.49, and 1.07, respectively. CIP was detected after all routes, but the AUC0-last ratios of CIP to ENR were <1.0% for plasma and muscle. ENR was detected up to 120 h following the IV, IP, or PO administrations. The long residence time of ENR after single IV, IP, or PO administration ensured the plasma concentration was ≥1 × MIC for bacteria with threshold MIC values of 0.92, 0.72, and 0.80 µg/mL over the whole 120 h observed. However, further studies are necessary to determine the optimum pharmacokinetic/pharmacodynamics data of ENR for the treatment of infections caused by susceptible bacteria in tilapia.

Pharmacokinetics of cefquinome in rainbow trout (Oncorhynchus mykiss) after intravascular, intraperitoneal, and oral administrations.

This study aimed to determine the pharmacokinetics and bioavailability of cefquinome in rainbow trout (Oncorhynchus mykiss) following intravascular (IV), intraperitoneal (IP), and oral (PO) administrations at 14 ± 1°C. In this study, three hundred and six clinically healthy rainbow trout (110-140 g) were used. The fish received single IV, IP, and PO injections of cefquinome at 10 mg/kg dose. The plasma concentrations of cefquinome were measured using HPLC-UV and were evaluated using non-compartmental analysis. Cefquinome was measured up to 96 h for PO route and 144 h for IV and IP routes in plasma. Following IV administration, t1/2ʎz , ClT , and Vdss were 18.85 h, 0.037 L/h/kg, and 0.84 L/kg, respectively. The Cmax of IP and PO routes was 9.75 and 1.64 µg/ml, respectively. The bioavailability following IP and PO administrations was 59.46% and 12.33%, respectively. Cefquinome at 10 mg/kg dose may maintain T > MIC above 40% at 72 and 96 h intervals, respectively, following the IP and IV routes for bacteria with MIC values of ≤2 µg/ml and at 24 h intervals following the PO route for bacteria with MIC value of ≤0.75 µg/ml. However, further studies are needed to determine in vitro and in vivo antibacterial efficacy and multiple dosage regimens of cefquinome against pathogens isolated from rainbow trout.

Effects of dietary marine sulphated polysaccharides (Algimun®) on growth performance, immune responses and disease resistance of juvenile gilthead seabream (Sparus aurata) to Photobacterium damselae subsp. piscicida.

The present study evaluated the effects of a dietary mix of marine sulphated polysaccharides, named Algimun® (AL), supplementation to gilthead seabream (Sparus aurata) juveniles in terms of growth performance, immune responses, and resistance against Photobacterium damselae subsp. piscicida. A total of 240 fish (initial mean weight of 6.00 ± 0.03 g) was randomly separated into 12 tanks (400 L, 20 fish per tank) distributed in four replicates. Fish were fed three experimental diets: a basal diet (Control), and a basal diet with two inclusion rates of Algimun® as 3 g/kg (AL0.3) and 5 g/kg (AL0.5) for 30 days before bacterial infection with P. damselae subsp. piscicida. After a 30-day feeding-period, growth performance was significantly improved in AL0.3 and AL0.5 groups compared to the control group (P < 0.05). AL0.3 and AL0.5 groups showed significantly higher lysozyme activity and myeloperoxidase activity when compared to the control group (P < 0.05). The gene expression of immune mediators (IL-1ß, IL-6, IL-10, IL-18, TNF-α and COX-2) was significantly upregulated in the intestine, spleen and head kidney in AL0.3 and AL0.5 groups when compared to the control group (P < 0.05). Eight days post-challenge, the survival rate against P. damselae subsp. piscicida was numerically higher in fish within AL0.3 and AL0.5 groups compared to control (+20%). The study findings suggest that marine sulphated polysaccharides (Algimun®) could be used as an immunomodulator in gilthead seabream to support animal's health and boost resistance in case of disease outbreak.

Pharmacokinetics and Pharmacokinetic/Pharmacodynamic Integration of Enrofloxacin Following Single Oral Administration of Different Doses in Brown Trout (Salmo trutta).

The pharmacokinetic of enrofloxacin was investigated in brown trout (Salmo trutta) following oral administration of 10, 20, and 40 mg/kg doses at 11 ± 1.5 °C. Furthermore, MICs of enrofloxacin against Aeromonas hydrophila and A. sobria were determined. The plasma concentrations of enrofloxacin and ciprofloxacin were determined using HPLC-UV and analyzed by non-compartmental method. Following oral administration at dose of 10 mg/kg, total clearance (CL/F), area under the concentration-time curve (AUC0-∞) and peak plasma concentrations (Cmax) were 41.32 mL/h/kg, 242.02 h*µg/mL and 4.63 µg/mL, respectively. When compared to 10 mg/kg dose, the dose-normalized AUC0-∞ and Cmax were increased by 56.30% and 30.08%, respectively, while CL/F decreased by 38.4% at 40 mg/kg dose, suggesting the non-linearity. Ciprofloxacin was not detected in the all of plasma samples. The MIC values of enrofloxacin were ranged 0.0625-4 µg/mL for A. hydrophila and 0.0625-2 µg/mL for A. sobria. The oral administration of enrofloxacin at 10 (for 192 h) and 20 (for 240 h) mg/kg doses provided the AUC of enrofloxacin equal to 1.23 and 1.96-fold MICs, respectively, for A. hydrophila and A. sobria with the MIC90 values of 1 µg/mL. However, further researches are needed on the PK/PD study of enrofloxacin for the successful treatment of infections caused by A. hydrophila and A. sobria in brown trout.

Modulatory effects of laurel-leaf cistus (Cistus laurifolius) ethanolic extract on innate immune responses and disease resistance in common carp (Cyprinus carpio).

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.

Antioxidant enzyme activities and immune responses in rainbow trout (Onchorhynchus mykiss) juveniles fed diets supplemented with dandelion (Taraxacum officinalis) and lichen (Usnea barbata) extracts.

In the present study, antioxidant activity, immune responses, and growth performance of rainbow trout (Onchorhynchus mykiss) juveniles fed with diets supplemented with dandelion (Taraxacum officinalis) and lichen (Usnea barbata) extracts were assessed. Four different concentrations of aqueous methanolic extract of the plants (0% (control), 0.1%, 0.5%, and 1% (D, dandelion; L, lichen) were added to the diets, and fish were fed for 75 days. On the 15th, 45th, and 75th day of the study, liver antioxidant enzyme activities were determined, and immune responses were determined every 15th day. The results showed that SOD activity increased in the fish group of 0.1% D on the 15th and 45th day compared to control; however, it was lower in all the lichen extract-treated groups than in control at almost all sampling times, except on the 15th day in the 0.1% L group. CAT activity showed an increased value (P < 0.05) in 0.5% L and 1% L treated fish groups on the 15th day, in fish of 1% D and 1% L groups on 45th and on 75th day in 0.1% D group. GPX activity increased on the 15th day of the study in fish of 0.1% D group, on the 45th day in 1% D and 1% L groups and on the 75th day in fish of 0.5% D, 0.1% D, and 0.5% L groups (P < 0.05). G6PDH enhanced in all treatment groups compared to control on the 15th day, except in 0.1% L and 0.5% L groups. An elevated G6PDH activity was also observed on the 75th day of the study in 0.5% D, 1% D, and 0.5% L fish groups. An increase on lipid peroxidation (LP) was observed in all L groups on the 45th day of the study. Lysozyme activity was determined to be the highest in 0.5% and 1% L on the 45th day, in 0.1% L on the 60th day and in the 0.5% L fish group on the 75th day compared to control (P < 0.05). Myeloperoxidase was found to be the highest at the end of the study in 1% L fish group compared to the control (P < 0.05). In conclusion, we suggest the use of dandelion to combat oxidative stress and to lower FCR and the use of lichen to modulate the immune response in rainbow trout. The use of such products will be economical for aquaculture and harmless for the environment.

A novel herbal immunostimulant for rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri.

In this 21-day study, we examined the effects of the aqueous methanolic extract of thin-skinned plum (Prunus domestica) on growth, immune response and resistance to a pathogenic bacterium, Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). Fish were fed with diets containing thin-skinned plum extract doses as 0 (Control) 0.1 (PD01), 0.5 (PD05) and 1% (PD1) ad libitum twice in a day. At the end of the study, growth was affected positively but not significantly. Feed conversion ratio (FCR) was decreased in the PD01 group (P < 0.05). There were elevated respiratory burst and potential bacterial killing activities on the 7th day in the PD1 fish group. No differences were observed in lysozyme activity (P > 0.05). An increased myeloperoxidase activity was recorded on the 14th day of study. Expression of interleukin and COX-2 genes was elevated on the 7th day of study in the kidney and intestine of treated fish. Histological results indicated no marked changes in organs (gill, kidney, liver and spleen) of PD treated fish groups. Challenge results of fish in all plum extract-treated groups showed an increased survival rate against Y. ruckeri (P < 0.05). This study indicated that the thin-skinned plum aqueous methanolic extract could improve innate immunity, survival against Y. ruckeri and decrease the FCR level.

Effects of Greek juniper (Juniperus excelsa) extract on immune responses and disease resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss).

This study investigated the effects of Greek juniper extract on immune responses of rainbow trout. In this experiment, 4 doses [0 (Control), 1 (J1), 4 (J4) and 8 (J8) mg/kg] of the extract were administered orally using an oral gavage twice a day for 14 days. Immune responses were measured on 7th and 14th days. On 14th day, Yersinia ruckeri was injected intraperitoneally to all fish of all groups. On 14th day, ORP in fish of J1 group increased significantly. Lysozyme activity (LA) was increased in J8 group on 7th day (p < .05). On 14th day, a significant decrease was determined in J1 and J4 treatments in LA. Myeloperoxidase activity was significantly decreased in all groups irrespective of sampling times (p < .05). Interleukin (IL)-1ß was significantly elevated in fish of J8 group on 7th day. IL-8 increased in fish of J8 and J4 groups on 7th day of the study. IL-12 gene expression was significantly up-regulated in J8 fish group on 7th day, and in J4 fish group on 14th day. Survival rate was higher in J8 treatment compared to the control and other treatments (p < .05). The results suggest that Juniperus excelsa provides protection against Y. ruckeri in rainbow trout.

Effects of ribwort plantain (Plantago lanceolata) extract on blood parameters, immune response, antioxidant enzyme activities, and growth performance in rainbow trout (Oncorhynchus mykiss).

In this study, we examined changes occurred in blood parameters, immune responses, antioxidant enzyme activities, and growth performance of rainbow trout (Oncorhynchus mykiss) administered with ribwort plantain (RP) through feed. Fish (mean weight 36.56 ± 1.99 g) were fed a diet supplemented with an aqueous methanolic extract of RP at variable doses, 0 (control), 1 (RP1), 2 (RP2), and 3 g kg-1 (RP3) for 90 days. The final weight, weight gain, and specific growth rate were significantly increased in RP1, RP2, and RP3 treatment groups compared to that of the control. Among examined blood parameters, hemoglobin value in RP1 group (9.77 ± 0.10 g dl-1) only was significantly high on the 30th day of the study. When immune response parameters were evaluated, we observed that oxidative radical production and lysozyme activities were affected positively in experimental groups (P < 0.05). The highest oxidative radical production was determined in fish of RP3 group. Glutathione peroxidase and glucose 6 phosphate dehydrogenase were increased in RP3 group compared to control and other treatment groups. Based on these results, it is concluded that ribwort plantain promotes growth, enhances immune responses and antioxidant enzyme activities in rainbow trout, and therefore, may be used in aquaculture.

Effect of lemon balm (Melissa officinalis) extract on growth performance, digestive and antioxidant enzyme activities, and immune responses in rainbow trout (Oncorhynchus mykiss).

This study was conducted to determine the effects of dietary supplementation of lemon balm (Melissa officinalis) aqueous methanolic extract on growth performance, blood parameters, digestive and antioxidant enzyme activities, and non-specific immune responses in rainbow trout (Oncorhynchus mykiss). Fish with an average weight of 23.03 ± 0.07 g were fed a diet supplemented with an aqueous methanolic extract of lemon balm at a dose of 0 (control), 0.1 (LB0.1), 0.5 (LB0.5), and 1 g kg-1 (LB1) for 75 days. The final weight, weight gain, and specific growth rate were observed to be significantly increased in LB0.5 and LB1 groups compared with that of the control. No differences were observed in feed conversion ratio values. WBC increased at the 45th day of the study in LB0.1 group. No differences were determined in RBC (P > 0.05). At the end of the study, lipase increased significantly in all experimental groups compared with the control. Pepsin was also elevated in LB0.5 and LB1 groups compared with the control. Increased trypsin was determined in LB1 group (P < 0.05). SOD activity increased at the end of the study in LB1 group (P < 0.05). CAT values had no differences compared with control. GR activity increased in all experimental groups compared with control. GPx improved in LB0.5 and LB1 groups significantly (P < 0.05). Lipid peroxidation was decreased in LB1 group compared with that of control, but this decrease was not significant (P < 0.05). Oxidative radical production and lysozyme activity significantly increased in LB1 group (P < 0.05). The highest MPO was determined in control group (P < 0.05). Current results suggest that lemon balm extract stimulates growth promoting antioxidant and immune responses in rainbow trout.

A new potential therapeutic remedy against Aeromonas hydrophila infection in rainbow trout (Oncorhynchus mykiss) using tetra, Cotinus coggygria.

Different antibiotic-based drugs are being used for the treatment of Aeromonas hydrophila infection in rainbow trout, and several studies emphasize the use of medicinal plants as immunostimulants for prophylactic measure against Aeromoniasis disease. However, therapeutic effects of aqueous methanolic extracts of tetra (Cotinus coggygria) against A. hydrophila in rainbow trout were not investigated. Four different concentrations of tetra extract (0 [control], 4, 8 and 12 mg/100 µl) and also two different positive control groups (florfenicol and doxycycline antibiotics) were administered orally using feeding needles to individual rainbow trout, Oncorhynchus mykiss of all experimental groups twice a day after intramuscular inoculation of A. hydrophila. The study period was for 10 days. On 0th, 3rd, 7th and 10th day, blood and tissues were collected from the fish and changes in humoral immune responses, haematology and immune-related gene expressions were determined. In the study, superoxide radical production was decreased generally in all experimental groups except in 12 mg tetra and florfenicol treatments compared to control (p < .05). Lysozyme activity was generally decreased (p < .05), or no differences were observed in all experimental groups compared to the control. Myeloperoxidase activity was significantly increased in florfenicol-treated fish group on 7th day (p < .05). Generally, myeloperoxidase activity showed an increase in almost all tetra-treated groups. Haematological parameters increased but were not significantly high enough in treatments. Almost all immune-related gene expressions were significantly enhanced on 3rd and 10th day of the study. Survival rate of 53.33% was found in control group. There were no significant differences in survival between control and 4 mg tetra-treated group (p > .05). All the other groups' survival rate was significantly increased compared to control. The highest survival rate was found in florfenicol group (80%). In 12 mg tetra-, doxycycline- and 8 mg tetra-treated groups, survival rate was recorded as 74.44%, 70% and 70%, respectively. Our results suggest that tetra methanolic extract is an effective therapeutic remedy against A. hydrophila infection in rainbow trout at the dose of 24 mg/32.34 g body weight/day.

Therapeutic effects of beard lichen, Usnea barbata extract against Lactococcus garvieae infection in rainbow trout (Oncorhynchus mykiss).

In this study, therapeutic effects of aqueous methanolic extracts of beard lichen (Usnea barbata) against Lactococcus garvieae (ATCC 43921) in rainbow trout (Onchorhynchus mykiss) were investigated. Six different experimental groups [0 mg/100 µL (Control), 4 mg/100 µL, 8 mg/100 µL, 12 mg/100 µL, 6 mg/100 µL florfenicol (positive control), 6 mg/100 µL erythromycin (positive control)] were set up to determine effects of lichen extract on immune responses and survival rate. In the study, superoxide radical production was increased in fish treated with 12 mg beard lichen extract, florfenicol and erythromycin compared to that of control (P < 0.05). Lysozyme activity was generally decreased (P < 0.05) or no differences were observed in all experimental groups compared to that of control (P > 0.05). Myeloperoxidase was significantly increased in all antibiotic treated groups. No differences were observed in liver histology of experimental groups compared to control. Cytokine gene expressions were elevated in all experimental groups compared to that of control (P < 0.05), except IL-1ß expression at 10th day sampling time. Other immune related genes (IL-8, TGF- ß, IL-12 Beta, TNFα1, IL-10, COX-2, IL-6, TLR5, C3, IGM, MHC-II, iNOS, IgT, IFN1, IFN2, IFN reg) were also elevated in all experimental groups compared to that of control group. The survival rates obtained in 4 mg beard lichen treated group, 8 mg beard lichen treated group and erythromycin treated group were 73.08, 65.38 and 80.77% respectively. Our results suggest that beard lichen methanolic extract could be an effective therapeutic agent to be used against L. garvieae infection in rainbow trout at the dose of 4 mg/17.41 ±â€¯0.3 g body weight/day.

Immune responses to methanolic extract of black cumin (Nigella sativa) in rainbow trout (Oncorhynchus mykiss).

The immune stimulating effects of the methanolic extract of black cumin (Nigella sativa) in rainbow trout (Oncorhynchus mykiss) was evaluated. Variable concentrations of black cumin methanolic extract [0 (Control), 0.1 and 0.5 g kg-1 of feed] were individually added to the basal diet and rainbow trout was fed for 30 days to assess the innate immune responses and growth performance. Feed conversion ratio significantly decreased in the group fed with 0.5 g kg-1 black cumin extract. Respiratory burst activity was observed to be the highest in the 0.5 g kg-1 black cumin extract fed group. Lysozyme and myeloperoxidase activities were significantly increased in fish of experimental groups compared to control (P < 0.05). TGF-ß gene expression increased in black cumin 0.5 g kg-1 treated group. IL-1ß and TGF-ß gene expressions decreased in black cumin 0.1 g kg-1 administered group. Expression of IL-12 gene diminished in both the experimental groups. There was no significant difference in survival rates between black cumin extract treated fish groups and control (P > 0.05) after challenged with Aeromonas hydrophila. The results indicate that the methanolic extract of black cumin is a stimulator of some innate humoral immune responses, but it is ineffective for cytokine-related gene trancriptions in rainbow trout.

Innate immune and growth promoting responses to caper (Capparis spinosa) extract in rainbow trout (Oncorhynchus mykiss).

Cytokine responses, non-specific immune activity and growth promotion effect of dietary caper (Capparis spinosa) supplementation were examined in rainbow trout (Oncorhynchus mykiss). Rainbow trout (12.04 ± 0.71 g) were fed diets containing three doses of caper methanolic extract [0 (Control), 0.1 and 0.5 g kg(-1) of feed] for 30 days. At the end of the feeding trial, expression levels of cytokine genes that included IL-1ß, IL-8, TGF-ß, IL-12p40, TNF-α1 and IL-10 in head kidney was analyzed using qRT-PCR, and blood and serum were collected to determine superoxide anion production (SAP), phagocytic, lysozyme and myeloperoxidase activities. Expression levels of all cytokines, except TNF-α1 were elevated in the 0.1 g kg(-1) caper extract fed fish group compared to other groups. In 0.5 g kg(-1) caper extract treated fish, only IL-12p40 and IL-10 genes were up-regulated compared to control group fish. SAP was increased in both caper extract treated groups compared to the control, and the highest level was observed in the 0.1 g kg(-1) group. Phagocytic activity in both the caper extract treated groups was increased compared to control with no differences observed between those groups. Lysozyme and myeloperoxidase activities were recorded to be the highest in the 0.1 g kg(-1) fed fish group compared to other groups. Growth promotion was affected positively when caper doses were increased. Survival rate was significantly higher in 0.1 and 0.5 g kg(-1) caper extract treated fish groups compared to control (P < 0.05) after challenged with Aeromonas hydrophila. These results indicate that caper extract stimulates innate immunity through cytokine-mediated responses and promote growth in rainbow trout.

Inflammatory immune response by lipopolysaccharide-responsive nucleotide binding oligomerization domain (NOD)-like receptors in the Japanese pufferfish (Takifugu rubripes).

Some of NOD-like receptors (NLRs), the cytosolic pattern recognition receptors form a multi-protein complex, inflammasome consisting of one or more NLRs, the adaptor protein ASC and inflammatory caspase to generate mature inflammatory cytokines, interleukin (IL)-1ß and IL-18. However, inflammasome-mediated inflammatory cascade involving any NLR member is unknown in a lower vertebrate like fish. Also, inflammatory cytokine induction pathway in response to a specific ligand, namely bacterial lipopolysaccharide (LPS) has not yet been clarified. Therefore, 13 predicted NLR sequences of the Japanese pufferfish, Fugu (Takifugu rubripes) were retrieved in silico and categorized as NLR-C1∼13. Expression analysis of these genes in Fugu head kidney (HK) cells stimulated with a heat-killed Lactobacillus paracasei spp. paracasei (Lpp), LPS, nigericin and a combination of nigericin + LPS showed consistent up-regulations of NLR-C1, 5, 7, 10 and 12 genes in both Lpp and LPS stimulations and NLR-C9 gene in LPS stimulation only. However, nigericin and nigericin + LPS caused an increased expression of NLR-C10 and 12 in HK cells and leukocytes. Fugu treated with Lpp and LPS (in vivo), and infected with Vibrio harveyi had an elevated expression of NLR-C10 and 12. Increased transcription of caspase-1, ASC, IL-1ß and IL-18 was recorded in nigericin-stimulated HK cells and leukocytes. Results suggested activation of probable inflammasome-mediated inflammatory cytokine response in Fugu. Moreover, LPS may be a key ligand that induces some of the Fugu NLR-Cs (NLR-C9, 10 and 12). Further characterization and functional analysis of Fugu NLR-C10 and 12 for ligand sensing, and processing of pro-inflammatory cytokine, IL-1ß would elucidate the inflammasome evolution in fish.

Growth performance and antioxidant enzyme activities in rainbow trout (Oncorhynchus mykiss) juveniles fed diets supplemented with sage, mint and thyme oils.

This study evaluated effects of dietary supplementation of sage (Salvia officinalis), mint (Mentha spicata) and thyme (Thymus vulgaris) oils on growth performance, lipid peroxidation level (melondialdehyde, MDA) and liver antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT; glucose-6-phosphate dehydrogenase, G6PD; glutathione reductase, GR; glutathione-S-transferase, GST and glutathione peroxidase, GPx) in rainbow trout (Oncorhynchus mykiss) juveniles. For this purpose, triplicate groups of rainbow trout were fed daily ad libitum with diets containing sage, mint and thyme oils at 500, 1,000 and 1,500 mg kg(-1) for 60 days. While weight gain percentage of fish fed the diets containing sage and thyme oils was significantly higher than the control group, that of fish fed mint oil was the lowest. Similarly, specific growth rate was found to be the highest in all groups of the sage and thyme oil feeding and the lowest in the mint groups. Moreover, feed conversion ratio was significantly higher in the mint oil administered groups. Survival rate was also significantly reduced in the fish fed the diet containing mint oil. It was observed that SOD, G6PD and GPx activities were significantly increased in liver tissues of all the treated fish groups compared to that of control diet-fed group. However, CAT, GST and GR activities were significantly decreased in experimental diet-fed fish groups at the end of the experiment. On the other hand, a significant reduction was found in MDA levels in the fish fed the diets with sage and thyme oils compared to control and mint diets on the 30th and 60th days of experiment. Overall, dietary inclusion of sage and thyme oils is effective in enhancing rainbow trout growth, reduction in MDA and least changing antioxidant enzyme activities at a low level of 500 mg kg(-1) diet, and they can be used as important feed supplements for rainbow trout production.