Supplementation of zebrafish (Danio rerio) diet using a short antimicrobial peptide: Evaluation of growth performance, immunomodulatory function, antioxidant activity, and disease resistance.

Short-chain bioactive peptides are new and promising antimicrobial, immune moderating, and antioxidant agents. Therefore, the present study was conducted to evaluate in vitro antibacterial activity of CM11, a short antimicrobial peptide (AMP), against Streptococcus iniae and Yersinia ruckeri as fish pathogenic bacteria using standard disk diffusion and microdilution assays. In addition, in vivo effects of CM11 on fish growth, immunity, antioxidant activity, and disease resistance were evaluated using zebrafish (Danio rerio) as an animal model. For in vivo study, based on in vitro susceptibility results, four diets were designed to include zero (as control), 10, 20, and 50 µg of CM11 per g diet referred to as control, P1, P2, and P3 treatments, respectively. After eight weeks of dietary trial, fish were challenged with Streptococcus iniae, and the survival rate was calculated for a period of two weeks. Results showed that CM11 effectively inhibited the growth of S. iniae and Y. ruckeri on agar plates at a concentration of eight µg/ml. Minimum inhibitory and minimum bactericidal concentrations of CM11 were measured at 8 and 32 µg/ml for S. iniae and 16 and 64 µg/ml Y. ruckeri, respectively. In vivo results showed no noticeable effects on fish growth parameters, however, feed conversion ratio (FCR) was found lower in P3 and P2 compared to control (P < 0.05). Immunological and antioxidant responses were found strongly affected by CM11 in all treatment groups in which the highest values were found in the P3 treated group. Key immune and antioxidant genes were up-regulated particularly in fish receiving the highest level of CM11 (P3). Fish receiving the CM11 peptide showed better survival when challenged with S. iniae. These findings suggest the potential of CM11 for use in aquaculture as an antibacterial and immunostimulant agent.

Evaluation of the efficacy of enrofloxacin in rainbow trout (Oncorhynchus mykiss) following experimental challenge with Yersinia ruckeri.

BACKGROUND: Use of enrofloxacin in trout farms is reported, especially for the treatment of yersiniosis, albeit various dosing regimens have been used. Therefore, optimal doses should be investigated. METHODS: Five groups of 15 fish were challenged with Y. ruckeri. Two days later, three groups received feed containing enrofloxacin (ENR) at 1, 2.5 and 5 mg/kg fish respectively, during 7 days; one group received a single intraperitoneal injection of ENR at 10 mg/kg; and one group was left untreated. On day 15, surviving fish were euthanized. RESULTS: All fish survived in the group treated by injection, compared to 53%, 60% and 40% of the fish treated with 1, 2.5 and 5 mg/kg oral ENR, respectively, and 53% in the infected untreated group. CONCLUSION: A single intraperitoneal injection of ENR at 10 mg/kg seems more relevant than repeated oral administrations. The ENR oral doses used in trout farms should be revised.

Dietary Chitosan Nanoparticles: Potential Role in Modulation of Rainbow Trout (Oncorhynchus mykiss) Antibacterial Defense and Intestinal Immunity against Enteric Redmouth Disease.

Bio-nanotechnology employing bio-sourced nanomaterial is an emerging avenue serving the field of fish medicine. Marine-sourced chitosan nanoparticles (CSNPs) is a well-known antimicrobial and immunomodulatory reagent with low or no harm side effects on fish or their human consumers. In this study, in vitro skin mucus and serum antibacterial activity assays along with intestinal histology, histochemical, and gene expression analyses were performed to evaluate the impact of dietary CSNPs (5 g kg-1 dry feed) on rainbow trout resistance against 'enteric redmouth' disease. Two treatment conditions were included; short-term prophylactic-regimen for 21 days before the bacterial challenge, and long-term therapeutic-regimen for 21 days before the challenge and extended for 28 days after the challenge. Our results revealed higher antibacterial defense ability and positive intestinal histochemical and molecular traits of rainbow trout after dietary CSNPs. The prophylactic-regimen improved trout health while the therapeutic regimen improved their disease resistance and lowered their morbidity. Therefore, it is anticipated that CSNPs is an effective antibacterial and immunomodulatory fish feed supplement against the infectious threats. However, the CSNPs seem to be more effective in the therapeutic application rather than being used for short-term prophylactic applications.

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 Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Extract of common mallow (Malvae sylvestris) enhances growth, immunity, and resistance of rainbow trout (Oncorhynchus mykiss) fingerlings against Yersinia ruckeri infection.

The dietary effects of a native medicinal plant from Iran, common mallow (Malvae sylvestris), was evaluated on growth performance, innate immune parameters, mucosal immune parameters, and resistance of rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri. Therefore, 360 fish (initial weight 10.42 ± 0.09 g) were randomly distributed into 12 fiberglass tanks. Experimental diets supplemented with 0 (as control- C), 1% (M1), 3% (M2) and 5% (M3) levels of M. sylvestris flowers extract were fed to the fish based on 3% of body weight for 8 weeks. At the terminal sampling, growth performance, liver and digestive enzymes activities, blood and mucosal immune responses were determined. Results showed that M2 and M3 had greater final weight, weight gain, SGR, survival rate and lower FCR; higher levels of total protein, albumin, globulin, and lower cortisol levels in comparison to control; 5% extract also lowered cholesterol and glucose levels as well as Lactate Dehydrogenase (LDH) activity. We reported higher values of hematocrit, hemoglobin, Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), White Blood Cell (WBC), Red Blood Cell (RBC) and lymphocytes for treated groups. Innate immune responses (Alternative complement activity (ACH50) in M2 and M3 group, total Immunoglobulin (Ig) and lysozyme in M3), mucosal immune parameters (ACH50, total Ig for M2 and M3 group and lysozyme in all treated groups) were enhanced. Activities of digestive enzymes (protease in all treated groups, amylase for 3 and 5%, while lipase only for 5%) and lower activity of liver ALT enzyme in individuals treated with highest dose was observed. Overall results indicated that the extract can positively affect growth performance and immune responses of rainbow trout.

Growth performance, haematological changes, immune response, antioxidant activity and disease resistance in rainbow trout (Oncorhynchus mykiss) fed diet supplemented with ellagic acid.

The present study was performed to investigate the effects of various levels of dietary ellagic acid (EA) on growth performance, haematological values, immune response, protection against Yersinia ruckeri infection, and oxidant/antioxidant status in rainbow trout, Oncorhynchus mykiss. Fish were fed with the control diet and three different experimental diets containing three graded levels of EA (50, 100 and 200 mg kg-1 diet) for 8 weeks. At the end of the experiment, the growth performance [weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR)], haematological values [the red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (Ht) level and erythrocyte indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)], immune response [white blood cell (WBC) count, oxidative radical production (nitroblue tetrazolium (NBT) assay), phagocytic activity (PA) and phagocytic index (PI), total protein (TP) and immunoglobulin M (IgM) levels, serum bactericidal activity (BA), lysozyme (LYZ) and myeloperoxidase (MPO) activities] and oxidant/antioxidant status [tissue malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities] were analysed. In addition, fish were challenged by Y. ruckeri and survival rate was recorded for 14 days. In the groups fed with EA the growth parameters such as WG, SGR, and FCR did not vary significantly. The RBC count, Hb concentration, and Ht level increased in the groups fed with EA when compared with the control group. However, there were no significant differences in the MCV, MCH and MCHC values among the groups. The results demonstrated enhancement in all the immunological parameters in the groups fed with EA compared to the control group. The results obtained from challenge with Y. ruckeri revealed reduction in the mortalities in the groups fed with EA. The dietary EA stimulated the SOD, CAT and GSH-Px activities in liver, head kidney and spleen as compared to the control group; however, a reverse trend was observed in the MDA levels of tissues. The present study suggest that EA can effectively enhance the haematological values, immune response, antioxidant capacity, and disease resistance in rainbow trout.

Protective effect of in-feed specific IgM towards Yersinia ruckeri in rainbow trout.

Tightened regulations and an environmentally friendly approaches in fish production have greatly reduced the use of antibiotics but green solutions are continuously being explored. The use of functional feed may have a potential in the aquaculture sector in securing biomass and minimizing the loss from disease. In the present study, we tested the concept that blood from the fish slaughterhouse can be used for mass purification of specific antibodies which subsequently can be used for feeding fish and thereby confer protection against diseases. IgM was purified from serum from Yersinia ruckeri vaccinated rainbow trout and an IgM sandwich ELISA was developed for quantification of rainbow trout IgM. The purified IgM was encapsulated in alginate microparticles and top-coated in fish feed. IgM re-extracted from the alginate microparticles was shown to retain high reactivity towards Y. ruckeri antigens indicating that its bioactivity remained intact after encapsulation. IgM release from the alginate microparticles was only observed at high pH (pH 8.2) and minimal at low pH, indicating protection of IgM at low pH in the fish stomach during passage. In a feeding - challenge experiment (feeding 1 week before Y. ruckeri challenge and for two weeks following challenge), a statistically non-significant 10% lower mortality was observed in the high dose (400 µg IgM/fish/day fed over 3 weeks) group.

Dietary effects of Coriandrum sativum extract on growth performance, physiological and innate immune responses and resistance of rainbow trout (Oncorhynchus mykiss) against Yersinia ruckeri.

This investigation was aimed to determine the efficacy of coriander seed extract (Coriandrum sativum) on physiological responses, immunity and disease resistance of rainbow trout, Oncorhynchus mykiss for eight weeks. A total number of six hundred rainbow trout (62 ±â€¯0.81 g) were divided into four feeding groups including 0 (control), 0.5%, 1% and 2% of coriander seed extract (CSE). In the present study, rainbow trout fed with 2% of CSE showed significantly higher values of specific growth rate (SGR), final weight (FW) and condition factor (CF) in comparison with control group after eight weeks (P < 0.05). Regarding hematological indices results, the 2% dosage of CSE showed the highest amount of hematocrit and hemoglobin compared to control group (P < 0.05). In addition, significant improvement of lysozyme and alternative complement activity, were observed in 2% of CSE treatment (P < 0.05). After eight weeks post-feeding, 30 fish from each treatment were challenged with Yersinia ruckeri for 14 days. The findings presented that fish fed with CES, especially 2% of CSE inclusion, improved survival rate of rainbow trout against Y. ruckeri; however, there were no significant differences among the fish in control and treatment groups at the end of the eight weeks feeding with coriander seed extract. The present study demonstrated, dietary incorporation of coriander extract can improve growth factors, immunological indices and resistance of rainbow trout (O. mykiss) against Y. ruckeri infection.

Possible effect of hala extract (Pandanus tectorius) on immune status, anti-tumour and resistance to Yersinia ruckeri infection in rainbow trout (Oncorhynchus mykiss).

The possible effect of dietary administration of hala extract (Pandanus tectorius) on rainbow trout (Oncorhynchus mykiss) immune status as well as its effect as an anti-tumour agent was studied. Fish were divided into 4 groups before feeding with commercial diet (0%, control; 0.5%, 1% and 2% of hala extract) for 2 weeks. The effect of diet on the humoral immune parameters, ie total protein, myeloperoxidase content, antiproteases, lysozyme and bactericidal activities were studied. Also, the effect of the diets on the expression of some immune-related genes in rainbow trout head-kidney (TNF, LYZ2, IL-8 and CD-4) as well as tumour suppressor gene (WT-1a) was investigated. At the end of the feeding trial fish groups were challenged with Yersinia ruckeri. The results demonstrated enhancement in all the immune parameters in fish fed hala extract diets compared to control fish especially with the highest dose (2%) which recorded the highest significant increase (p < 0.05) in some parameters (total protein, myeloperoxidase content, antiproteases, and bactericidal activities) compared to the control. The results obtained from challenge with Y. ruckeri revealed reduction in the mortalities in fish groups fed with 1% and 2% doses of hala extract. Feeding with hala extract provoked upregulation in all immune- related genes. Again, the highest dose of hala extract showed a significant upregulation in WT1a expression (p < 0.05). The current study suggest that the hala extract, especially the highest dose, could be considered a good food additive to improve the immune status, resist tumour formation and to resist or control infectious diseases of rainbow trout.

Effect of immunostimulatory feed supplements on the development of acquired immunity in rainbow trout (Oncorhynchus mykiss).

Immunostimulatory feed supplements are an increasingly common feature of aquaculture management and their benefit has been confirmed for a wide area of products. However, these investigations have often focused on the benefit of these supplements on the innate immune system. In the current project, we investigated a mixture of two commercial feed supplements (Biotronic® Top 3 and Levabon® Aquagrow E) with a known protective effect against bacterial infections. The effect of the supplemented diet on antibody titters of Oncorhynchus mykiss vaccinated against Yersinia ruckeri was determined by ELISA. Furthermore, an infection trial was performed to confirm the effect of the supplements on the survival of the fish. Finally, their effects on the growth parameters of the fish were also determined. The results from this study found no significant effect on the general antibody titters. However, when considering only the titters of specific anti-Y ruckeri antibodies, the supplemented feed was associated with an improved response to the vaccine, significantly better than in the fish that had received the control feed.

The effects of common yarrow (Achillea millefolium Linnaeus), cinnamon (Cinnamomum zeylanicum Blume) and rosemary (Rosemarinus officinalis Linnaeus) hydrosols on the some immunological and hematological parameters of common carp (Cyprinus carpio L., 1758).

In this study, the effects of some plant hydrosols (distilled plant waters) based upon some hematological parameters and Nitroblue Tetrazolium (NBT) activities in the common carp (Cyprinus carpio Linnaeus, 1758) infected with Yersinia ruckeri were investigated. In the trial, it was utilized totally 200 common carps with 54.3±6.7 g mean live weight and 15.7±1.8 cm mean total lenght. The 10% rate of the common yarrow (Achillea millefolium Linnaeus) hydrosol; 0.5% rate of the cinnamon (Cinnamomum zeylanicum Blume) hydrosol; and 5% rate of the rosemary (Rosemarinus officinalis Linnaeus) hydrosol were applied to fish as a bath treatment. The erythrocyte (RBC), leukocyte count (WBC), hematocrit value (HCT), haemoglobin amount (Hg), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and activities of NBT in the blood samples taken from the caudal vena of the control and experimental fish groups were analyzed in the 7th, 14th, and 21st days of the exposure treatment. At the end of the research, HCT, Hg, RBC, WBC, MCH and MCV values decreased in the C-2 Group (the control group contain pathogen) compared to the C-1 Group (the control group no contain pathogen), except MCHC value. The NBT activities in the C-1 Groups increased at the 14th day, but decreased quite a few at the 21st day. It has been consequently reached the conclusion that the bath treatments of the some plant hydrosols might be beneficial in increasing of antibacterial properties and in strengthening of defense mechanisms of common carp against Y. ruckeri pathogen.

Trans-cinnamic acid application for rainbow trout (Oncorhynchus mykiss): I. Effects on haematological, serum biochemical, non-specific immune and head kidney gene expression responses.

The present study investigated the effects of dietary trans-cinnamic acid (CA) on pre- and post-challenge haematological, serum biochemical, non-specific immune and head kidney gene expression responses of rainbow trout, Oncorhynchus mykiss juveniles. In this regard, fish with an average weight of 17.01 ±â€¯0.05 g were divided into five groups, and fed daily with an additive free basal diet (control); 250, 500, 750 or 1500 mg kg-1 CA for a 60-day period. Fish were sampled every 20 days during the experiment. On days 20, 40 and 60 (the pre-challenge period), the dietary CA especially at 250 and/or 500 mg kg-1 significantly increased blood granulocyte percentage, and serum total protein, globulin, lysozyme and total immunoglobulin values. Furthermore, dietary CA increased activities of phagocytic activity, respiratory burst and potential killing, and increased the expression levels of immune related genes [serum amyloid A (SAA), interleukin 8 (IL-8), interleukin 1, beta (IL-1ß), transforming growth factor beta (TGF-ß), tumor necrosis factor (TNF-α), and immunoglobulin T (IgT)] in the head kidney of fish fed with 250 and/or 500 mg kg-1 CA. Following 60 days of feeding, fish were challenged with Yersinia ruckeri and mortality was recorded for 20 days. Highest percentage survival (%) rate was found in the 250 and/or 500 mg kg-1 CA-supplemented feeding groups. During the post-challenge period, red blood cell (RBC) count, hematocrit (%), respiratory burst activity, and total antiprotease activity increased in fish fed with feed containing 500 mg kg-1 content. Moreover, markedly up-regulated the expression of related genes (SAA, IL-8, IL-1ß, TGF-ß, TNF-α, IFN-γ and IgM) in fish fed 250, 500 and/or 750 mg kg-1 CA. Therefore, feeding O. mykiss for 60 days with dietary CA at 250-500 mg kg-1 CA incorporation levels can be suggested as optimal to enhance the immunity and disease resistance against Y. ruckeri.

Immunological responses and disease resistance of rainbow trout (Oncorhynchus mykiss) juveniles following dietary administration of stinging nettle (Urtica dioica).

The present study investigated the effects of dietary supplementation of stinging nettle (Urtica dioica) on growth performance, skin mucus, immune response and disease resistance of rainbow trout (Oncorhynchus mykiss) fed with diets supplemented with U. dioica at 0, 1, 2 and 3%. After 8 weeks of feeding, the addition of U. dioica at 3% level resulted in improved weight gain, specific growth rate and feed conversion ratio significantly when compared to the other groups (P < 0.05). Hematological responses including: hematocrit (Htc), hemoglobin (Hb), lymphocyte and neutrophil populations enhanced significantly in fish fed 3% of stinging nettle when measured after 4 weeks; while, total red blood cells, white blood, Htc, Hb, lymphocyte and neutrophil populations significantly increased after 8 weeks in the same group (P < 0.05). Total serum protein and glucose contents increased significantly in fish fed stinging nettle at 3% when compared to the other groups after 8 weeks; however, triglycerides decreased significantly in the same group on the 4th and 8th week (P < 0.05). Additionally, several immune parameters, namely, IgM, lysozyme, complement components C3 and C4, and respiratory burst of blood leukocytes significantly increased in the 3% fed group on the 4th week; while, after 8 weeks the immune responses enhanced in fish fed 2 and 3% diets (P < 0.05). At the end of the feeding trial, mucus samples obtained from the fish fed stinging nettle supplementation exhibited improved antagonistic activities against several bacterial pathogens (Streptococcus iniae, Yersinia ruckeri, Vibrio anguillarum and Lactococcus garviae), skin mucus enzymes activities (alkaline phosphatase, lysozyme, protease and esterase) and protein levels in 2 and 3% groups with the highest being in case of 3% group when compared to the other groups (P < 0.05). The cumulative mortality of rainbow trout subjected to Y. ruckeri infectious exhibited relatively low mortality levels in all supplemented groups with the lowest being in fish fed 3% stinging nettle. The present findings demonstrated that dietary administration of U. dioica enhanced growth and stimulated fish immunity; thus, enabling the fish to be more resistant against bacterial infections.

Hemato - Immunological and biochemical parameters, skin antibacterial activity, and survival in rainbow trout (Oncorhynchus mykiss) following the diet supplemented with Mentha piperita against Yersinia ruckeri.

This study was aimed to assess the potential effects of Mentha piperita on the hemato - immunological and biochemical parameters, skin antibacterial activity and protection against Yersinia ruckeri infection in rainbow trout Oncorhynchus mykiss. Fish were divided into 4 groups before being fed diets supplemented with 0, 1, 2 and 3% of Mentha piperita (MP) plant extract for 8 weeks. Dose-dependent increases immune (both in skin mucus and blood serum) and hematological parameters (number of red and white cells, hematocrit and hemoglobin contents), as well as in respiratory burst activity, total protein, albumin, and neutrophil levels in fish fed supplemented diets compared to the control fish. Furthermore, dietary MP plant extract supplements have no significant effect on blood biochemical parameters and enzymatic activities of liver determined in serum of rainbow trout. After 8 weeks the cessation of feeding with MP plant extract, survival rates of 54.4%, 63.6% and 75.2% were recorded in groups which received 1, 2 and 3% of MP plant extract of feed, respectively, compared to 34.6% survivals in the control. This study underlying several positive effects of dietary administration of MP plant extract to farmed fish.

Improvement of immunity and disease resistance in the Nile tilapia, Oreochromis niloticus, by dietary supplementation with Bacillus amyloliquefaciens.

Probiotics can be used as immunostimulants in aquaculture. The aim of this study was to evaluate the immune responses of Nile tilapia Oreochromis niloticus following feeding with Bacillus amyloliquefaciens spores at concentrations of 1 × 10(6) (G3) and 1 × 10(4) (G2) colony-forming units per gram (CFU/g) of feed compared with a basal diet with no probiotics (G1). A total of 180 fingerlings (27.7 ± 0.22 g) were divided into three groups (G1-G3 of 20 fish per group) in triplicate. Innate immunities were measured every two weeks based on serum bactericidal activity, lysozyme activity, a nitric oxide assay (mmo/l) and phagocytic activity, and the expressions of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF α) were examined after one month. Moreover, the survival of tilapia upon challenge with Yersinia ruckeri or Clostridium perfringens type D was determined at the end of feeding trial. After 15 d, the serum killing percentages and phagocytic activities were significantly higher in G3 than in G1 and G2, whereas the same parameters had significantly higher values in G3 and G2 than in G1 after 30 d. After both 15 d and 30 d, the lysozyme activities and nitric oxide assay results (mmo/l) were significantly higher in G3 than G2, and the lowest values were observed in G1. The percentage of serum killing, serum nitric oxide and serum lysozyme activity were significantly increased by the time of B. amyloliquefaciens administration independently of the probiotic dose, and the phagocytic activity percentage was significantly decreased at the end of the experiment. Dietary B. amyloliquefaciens caused significant increases in IL-1 and TNF α mRNA levels in the kidneys in the following pattern: G3 > G2 > G1. Fish that were fed B. amyloliquefaciens exhibited better relative survival percentages than the controls when challenged by Y. ruckeri or C. perfringens type D. Dietary supplementation with B. amyloliquefaciens improves immune status and disease resistance in Nile tilapia.