Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract.

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg-1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Stimulatory effect of dietary chasteberry (Vitex agnus-castus) extract on immunity, some immune-related gene expression, and resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus).

Chasteberry is highly recommended as an herbal medicine across the globe for treating of many gynaecological disorders. In this study, chasteberry extract (CBE) was supplemented in goldfish diet to evaluate the immunity responses at the cellular and molecular levels. Moreover, after the feeding trial, the fish were challenged with Aeromonas hydrophila. The fish (300 individuals, 2.4 ± 0.12 g initial weight) were randomly distributed in 12 tanks and were fed with 0 (control), 5, 10, and 15 g CBE per kg of feed for 8 weeks. Based on the results, lysozyme activity, alkaline phosphatase, and total immunoglobulin (Ig) in the skin mucus samples were significantly enhanced in the fish fed with 15 g/kg CBE (P < 0.05). Moreover, dietary CBE positively affected lysozyme activity, complement components, and IgM content of the serum samples compared to the control group. Also, the number of monocytes and lymphocytes were increased significantly with increasing CBE in the diet (P < 0.05). The highest mRNA levels of tumor necrosis factors (TNF-α, TNF-2α) and Lysozyme were observed in 15 g/kg CBE treatment. After the challenge test, the highest relative percentage survival value (60%) was observed in the fish fed with 15 g/kg CBE. We concluded that dietary CBE especially at 15 g/kg has an immunomodulatory effect in goldfish by stimulating the innate immunity and some inflammatory cytokines as well as disease resistance against A. hydrophila.

Effects of dietary fish meal replacement by fermented moringa (Moringa oleifera Lam.) leaves on growth performance, nonspecific immunity and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III).

This study was aimed to evaluate the effects of partial replacement of fish meal by fermented moringa leaves (FMLs) on growth performance, serum biochemistry, antioxidant status, nonspecific immunity, and resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III). Four isonitrogenous and isoenergetic balanced diets, including three FML diets (substituting 20%, 40%, 60% of the fish meal in basal diet, F20, F40 and F60, respectively) and a basal diet (a diet containing 10% fish meal) were used. Each diet was randomly allocated to four fish groups (F20, F40, F60 and control) reared in a recirculating system. After 50 days of the feeding trial, fish were challenged by A. hydrophila. The result revealed that final mean body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) were significantly increased (P < 0.05) in F20 and F40 groups compared with the control group. Decreased hepatosomatic index (HSI), body crude lipid, serum aspartate transaminase (AST) and serum alanine aminotransferase (ALT) activities, and increased serum alkaline phosphatase (AKP) and serum glutathione peroxidase (GPx) activities were observed in F40 and F60 groups compared with the control and F20 groups. All FMLs-supplemented groups increased (P < 0.05) serum superoxide dismutase (SOD), catalase (CAT) and lysozyme activities, complement component 3 (C3) and serum immunoglobulin M (IgM) concentration, or decreased serum malondialdehyde (MDA) and protein carbonyl (PCC) contents (P < 0.05). After the challenge test, the significant downregulation of toll-like receptors2 (TLR2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-8 mRNA transcription levels was observed in spleens of FMLs supplemented groups. Dietary F40 and F60 showed higher (P < 0.05) relative percent survival (RPS) (48.72% and 43.59%, respectively) against A. hydrophila infection than control. These results indicate that, as a dietary fish meal substitute, FMLs enhance the growth, and antioxidant and immune response, and regulate the expression of immune-related genes and increase disease resistance against A. hydrophila via TLR2 pathway in gibel carp, with greatest effects of 40% fish meal substitution.

Dietary supplementation of Geotrichum candidum improves growth, gut microbiota, immune-related gene expression and disease resistance in gibel carp CAS â ¢ (Carassius auratus gibelio).

Geotrichum candidum is a filamentous fungus mostly used in the dairy industry for cheese ripening and flavoring. The current study was designed to evaluate the potential probiotics effect of dietary supplementation of G. candidum on growth, digestive enzymes activities, gut microbiota and immune-related gene expression and disease resistance in Gibel carp CAS Ⅲ (Carassius auratus gibelio) against A. hydrophila infections. For this purpose, 420 healthy fish (initial body weight: 7.09 ± 0.02g) were randomly divided into 7 feeding groups in triplicates of 20 fish in each tank. Seven isocaloric and isonitrogenous diets were prepared, supplemented with 6 different doses of G. candidum viz; C: 0, T1:106, T2:107, T3:108, T4:109, T5:1010, T6:1011 CFU/kg diet and fed to fish twice a day for 60 days. Results showed significantly higher weight gain (WG) and feed efficiency (FE) in groups fed 106 and 108 CFU/kg diet compared to the control (P < 0.05). All G. candidum fed groups showed higher intestinal α-amylase activity (P < 0.05) while no difference in lipase and trypsin activity compared to the control group. Before challenge test, gut microbial diversity analysis revealed relatively more abundance of bacteria belonging to phylum Proteobacteria, Actinobacteria, Firmicutes and Bacteriodetes in group fed G. candidum supplemented diet and higher percentage of pathogenic bacteria belonging to order Aeromonadales and Vibrionales in control post-challenge. After challenge test immune response expressed numerically higher survival rate (P > 0.05) and significantly higher (P < 0.05) Respiratory Burst Activity(RBA), Immunoglobulin M level (IgM), Aspartate Transaminase activity (AST) and improved (P > 0.05) lysozyme activity, and Alanine Transaminase activity (ALT) in groups fed 106 and 108 CFU/kg G. candidum supplemented diet. The expression of immune related gene (IL-I ß, TNF- α, HSP70 and TLR-2) in liver of the fish were significantly affected (P < 0.05) by supplementation of G. candidum post bacterial challenge. The results showed that G. candidum is a potential probiotic as it could improve feed utilization, immunity and cause no harmful effects on growth of gibel carp at higher supplementation levels. The productive results can be achieved by using the lower dose as 106-108 CFU/kg diet.

Poly-ß-hydroxybutyrate (PHB) in bioflocs alters intestinal microbial community structure, immune-related gene expression and early Cyprinid herpesvirus 2 replication in gibel carp (Carassius auratus gibelio).

The aquaculture system based on biofloc technology (BFT) showed positive effects on prevention of Cyprinid herpesvirus 2 (CyHV-2) infection in gibel carp (Carassius auratus gibelio), which is detrimental to health and causes seriously economic losses to aquaculture. However, the enhancement mechanism of BFT regarding immunity and disease resistance of cultured species is scarce. Poly-ß-hydroxybutyrate (PHB) has been proved as one of bioactive compounds in bioflocs. In this study, two groups (4% PHB supplementation diets and control with basal diets) with 30-day feeding were set to study the effect of PHB supplementation on immune-related gene expression by qRT-PCR, time-course CyHV-2 replication in vivo by qPCR and intestinal microbiota by illumine high-throughput sequencing. PHB supplementation significantly up-regulated transcriptional levels of eight immune-related genes, decreased cumulative mortality of gibel carp and early CyHV-2 replication in spleen in vivo (P < 0.05). Additionally, PHB changed the microbial structure but not diversity, and significantly increased beneficial bacteria such as Bacillus sp. KEGG pathway analysis by PICRUSt demonstrated that oral administration of PHB up-regulated abundances of genes responsible for seven pathways and down-regulated genes in eleven pathways. Histological structures of foregut, mindgut and hindgut were also affected. Our findings suggested that profitable effects of PHB on immunity and disease resistance might be gut microbiota-related, and regulated through pathways of enzymes secretion, replication and repair, and host immune system. This study will provide new insights into understanding the enhancing mechanism of BFT on immunity and disease resistance of cultured animals, and developing prebiotics/probiotics-based immunotherapies to improve animal health and disease resistance.

The roles of two myostatins and immune effects after inhibition in Qi river crucian carp (Carassius auratus).

Myostatin, through type I receptor (kinase 4, 5, ALK4/5), functions to participate in the immune system and negatively regulate muscle growth in mammals. However, the role of myostatin (mstn) in the immune system of teleosts is largely unknown. In a previous study, we cloned the mstn1 cDNA encoding myostatin in Qi river crucian carp (Carassius auratus). In the present study, we have cloned mstn2 cDNA, which was characterized and analyzed together with mstn1. Tissue distribution analysis showed that both mstn genes are expressed in numerous tissues, with mstn1 dominantly expressed in the muscle and brain, whereas mstn2 is mainly expressed in the brain. During embryogenesis, mstn1 and mstn2 exhibit different expression patterns. Both mstn1 and mstn2 expression increased stepwise in the brain at different developmental stages. Furthermore, both genes are differentially regulated during different periods of fasting/re-feeding. Following the exposure of C. auratus to polyI:C, lipopolysaccharide (LPS), and Aeromonas hydrophila, both genes were upregulated in different tissues, which indicated that they might be involved in the immune response against pathogenic invasion. Blocking the Mstn signal pathway with SB-431542 (a chemical inhibitor of ALK4/5) resulted in significantly increased body length and weight. However, the mortality of SB-431542-treated fish was higher after A. hydrophila challenge. Moreover, decreased expression of lysozymes (lyz), complement component 3 (c3), ß-defensin 3 (defb3), and interferon γ (ifnγ) were exhibited in treated fish, compared with the controls. Furthermore, the expression of nf-κb1, three pro-inflammatory cytokines (il1ß, il6, and tnfα), and inflammatory cytokines (il8 and il10) were significantly increased in both the SB-431542-treated group and the control after A. hydrophila infection, suggesting that the NF-κB pathway was not suppressed in the SB-431542-treated fish. Taken together, our data suggest that both mstn1 and mstn2 play important roles in early body development, muscle growth, and the immune system by acting downstream of the NF-κB signal pathway.

Effects of inosine 5'-monophosphate supplementation in high fishmeal and high soybean diets on growth, immune-related gene expression in gibel carp (Carassius auratus gibelio var. CAS â ¢), and its challenge against Aeromonas hydrophila infection.

The present study was conducted to evaluate dietary inosine 5'-monophosphate (5'-IMP) on growth, immune genes expression and disease resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS Ⅲ) (initial body weight: 7.48 g). Six diets were formulated containing exogenous 5'-IMP at three gradient levels (0, 0.1% and 0.2%) in the high dietary fishmeal group (15% fishmeal: D1, D2, D3) and in the high dietary soybean meal group (33% soybean meal: D4, D5, D6). Each diet was randomly allotted to triplicate tanks in a recirculating system. After the feeding trial, fish were exposed to Aeromonas hydrophila challenge. Hematological and immunological responses were analyzed before and after challenge. The results indicated that feeding rate in all 5'-IMP supplemented treatments (D2, D3, D5 and D6) and daily growth coefficient in D5 and D6 were reduced compared with those of respective control treatments (D1 and D4) without 5'-IMP addition (P < 0.05). The cumulative survival rates were numerically improved by dietary 5'-IMP supplementation (P > 0.05). Compared with the respective control treatment, in the high fishmeal group, plasma SOD and MPO were significantly elevated in D3 at the end of feeding trial (P < 0.05), plasma SOD and lysozyme were significantly increased in D3 after bacterial challenge (P < 0.05); in high soybean meal group, plasma lysozyme activity was significantly elevated in D5 post bacterial challenge (P < 0.05). Most of the expression of immune related genes (intelectin, major histocompatibility complex class II ß (MHC II ß), Complement 3 (C3), Complement component C7-1 (ccC7), lysozyme C, Interleukin 1ß (IL-1ß), Tumor necrosis factor α1 (TNF-α1), Transforming growth factor-beta (TGF-ß) and Interleukin 8 (IL-8)) in spleen, kidney and liver of the fish were significantly affected by supplementation of 5'-IMP at the end of feeding trial and post bacterial challenge. Additionally, adding 5'-IMP in high soybean meal diets exerted further effects of promoting immunity than counterparts in high fishmeal diets. Considering enhanced disease resistance, the immunopotentiation of 5'-IMP was manifested when the addition level was 0.1% in high soybean meal diets and 0.2% in high fishmeal diets.

Replacement of fishmeal by spirulina Arthrospira platensis affects growth, immune related-gene expression in gibel carp (Carassius auratus gibelio var. CAS III), and its challenge against Aeromonas hydrophila infection.

The present study examined the effect of dietary spirulina, Arthrospira platensis on growth performance, blood physiological indices, immune-related gene expressions and resistance of juvenile gibel carp against Aeromonas hydrophila infection. Four isonitrogenous (360 g kg-1) and isolipidic (90 g kg-1) diets were formulated with containing different levels of spirulina powder of 0 g (SP0, the control diet), 3.38 g (SP3.38), 6.76 g (SP6.76) and 13.52 g (SP13.52) per 100 g diet to replace 0%, 25%, 50% and 100% of fishmeal protein, respectively. And each diet was randomly assigned to triplicate tanks (150-L capacity per each) and each tank was stocked with 22 fish (15.37 ±â€¯0.06 g). Fish were fed one of the tested diets up to satiation twice a day for 46 days. A challenge test was carried out after the feeding trial by injecting Aeromonas hydrophila intraperitoneally for 7 days. The results showed that fish growth, feeding rate in groups SP3.38 and SP6.76 were significantly higher than those of groups SP0 and SP13.52 (P < 0.05). Feed efficiency and protein retention rate had no significant difference among all tested groups. Plasma superoxide dismutase and phagocyte activity of blood leukocytes significantly increased in the spirulina-fed fish groups at 12-h post the bacterial challenge (P < 0.05). Both pre and post challenge test, plasma lysozyme activities in spirulina-fed groups were significantly higher than that in the control group (P < 0.05). Plasma malondialdehyde got the lowest value in the SP13.52 group before and after the challenge test. The transcriptional levels of TLR2 (Toll like receptor 2), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), interleukin-1ß (IL-1ß) and tumor necrosis factor-α1 (TNF-α1) in spleen and kidney significantly increased post the bacterial challenge compared to the pre challenge. And the relative expressions of the immune-related genes of spirulina-fed fish groups were higher than those of the control group before and after the challenge test. The 7-day cumulative survival rate after the bacterial challenge was highest in the SP3.38 group (P < 0.05). The present results indicated that low dietary inclusion of spirulina significantly enhanced the immune response of gibel carp partly through TLR2 pathway and 3.38% of dietary spirulina was recommended for the juveniles based on the growth and immune response.

Combined or Individual Effects of Dietary Probiotic Pedicoccus acidilactici and Nucleotide on Growth Performance, Intestinal Microbiota, Hemato-biochemical Parameters, and Innate Immune Response in Goldfish (Carassius auratus).

This study investigated the effect of dietary supplementation of probiotic Pedicoccus acidilactici and nucleotide (separately or combined) on growth performance, intestinal microbiota, hemato-immunological parameters, and immunity response in goldfish (Carassius auratus). Fish (average weight 5-6 g) were acclimatized and divided into eight experimental diets supplemented with P. acidilactici of different concentrations (0.1, 0.2, and 0.3% diet) and nucleotides (0 and 0.5% diet) for 6 months. Fish fed with experimental diets showed significant differences in terms of final weight, weight gain, feed conversion ratio, daily growth rate, and condition factor when compared to control diet (P < 0.05). Fish fed with probiotic (0.3%) separately and combined with nucleotide (0.5%) had highest RBC and WBC when compared to other diets (P < 0.05), while the highest values for Hb and Hct as well as total protein, glucose, albumin, and globulin were observed in probiotic (0.2%) and nucleotide (0.5%) combined diet. Serum lysozyme and anti-protease activities were significantly higher in probiotic (0.1 and 0.2%) and nucleotide (0.5%) combined diets. Similarly, these two diets combined showed the highest colonization of P. acidilactici when compared to other diets. In conclusion, combined dietary probiotic and nucleotide improve the growth performance, hemato-biochemical parameters, and intestine growth in C. auratus.

The effects of galactooligosaccharide on systemic and mucosal immune response, growth performance and appetite related gene transcript in goldfish (Carassius auratus gibelio).

The present study investigates the effects of supplementation of goldfish (Carassius auratus gibelio) diet with galactooligosaccharide (GOS) on serum immune response, mucosal immune parameters as well as appetite-related (Ghrelin) and immune-related (TNF-1α and TNF-2α) genes expression. One hundred and eighty fish with an average weight of 4.88 ± 0.28 g were stocked in twelve 500-L fiberglass tank assigned to four treatments repeated in triplicates. Fish were fed on experimental diets contain 0.5, 1 and 2% GOS for 6 weeks. Supplementation of diet with GOS had no remarkable effect on goldfish growth performance (P > 0.05). Evaluation of serum innate immune parameters revealed that supplementation of diet with GOS significantly elevated total protein, Albumin, Globulins, Lysozyme and Alkaline phosphatase activity as well as agglutination compared to control group in a dose dependent manner (P < 0.0.5). Also, Fish fed 2% GOS supplemented diet showed increased skin mucus immune response (total protein and lysozyme activity) compared other groups (P < 0.0.5); except in case of ALP activity. Molecular studies on appetite (ghrelin) and inflammatory cytokine (TNF-1α and TNF-2α) genes expression revealed remarkably decrease and increase, respectively in GOS fed fish (P < 0.0.5). These results showed immunomodulatory effects of dietary GOS on serum and skin mucus response as well as expression of inflammatory cytokines in goldfish, though this supplement decreased appetite gene expression and had no effect on growth performance.

Herbal supplementation diets on hematology and innate immunity in goldfish against Aeromonas hydrophila.

Goldfish, Carassius auratus (47 + or - 3 g, n = 300) were inoculated intramuscularly (50 microl) with Aeromonas hydrophila (1.8 x 10(6) cells ml(-1)). On the 6th day of post-infection the fishes were divided into i) control, without infection fed with normal diet (C), ii) infected fish, fed with normal diet (IU), and infected fishes treated with different doses of iii) 100 mg kg(-1), iv) 200 mg kg(-1), iv) 400 mg kg(-1) and vi) 800 mg kg(-1) mixed herbal extracts supplementation diets. Hematological and immunological parameters were determined on week 1, 2 and 4. In infected goldfish were fed diets containing 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds, the white blood cell (WBC) levels significantly increased (P < 0.05) throughout the experimental trial compared to the control. During the experimental period, the red blood cell (RBC) and haemoglobin (Hb) level in goldfish significantly decreased (P < 0.05) when fish fed with 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds while it was restored near control when infected fish fed with 400 or 800 mg kg(-1) of herbal extracts supplementation feeds. On the other hand, the haematocrit (Ht) values decline significantly (P < 0.05) in 100, 200 and 400 mg kg(-1) of mixed herbal supplementation feeding groups on weeks 2 and 4 when compared to control group. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) values almost significantly differ from the control values. The infected goldfish and treated with 100 or 200 mg kg(-1) of herbal supplementation feeds exhibited significantly decline (P < 0.05) in total protein (TP), glucose (GLU) and cholesterol (CHO) levels on week 1-4 whereas it was restored when infected fish fed with 400 or 800 mg kg(-1) of herbal supplementation feeds on week 4. In comparison to untreated control goldfish, the respiratory burst activity and phagocytic activity of blood cells was significantly enhanced in infected fish feeding with 200, 400 and 800 mg kg(-1) of herbal supplementation feeds compared to the control. On the other hand, infected fish fed with all the doses of mixed herbal supplementation feeds, the lysozyme activity was significantly enhanced throughout the experimental period. This study shows that the infected goldfish treated with 400 and 800 mg kg(-1) of herbal supplementation feeds preceding the challenge with live A. hydrophila had 30% and 25% mortality. However, 100 and 200 mg kg(-1) of herbal supplementation feeds treated groups were found the percentage mortality 50% and 45%, respectively. Our results indicate that 400 or 800 mg kg(-1) of mixed herbal supplementation feeds were restored the altered hematological parameters and triggering the innate immune system of goldfish against A. hydrophila.

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts.

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg(-1) body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 x 10(7) cells ml(-1)). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk.

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.