Enhancement of protective immunity in blue gourami,Trichogaster trichopterus (Pallas), against Aeromonas hydrophila and Vibrioanguillarum by A. hydrophila major adhesin

Blue gourami, Trichogaster trichopterus (Pallas), were intraperitoneally immunized with major adhesin, a 43 kDa OMP protein isolated from fish Aeromonas hydrophila, in the presence of Freund's complete adjuvant (FCA). Three weeks later, a booster injection of adhesin without FCA was administered. Control group fish were similarly treated with phosphate‐buffered saline (PBS) and FCA. Results showed that anti‐adhesin serum obtained from fish after booster immunization exhibited very strong ability in agglutinating bacterial cells. Although this antiserum had no bactericidal effect, it could significantly inhibit serologically different strains of A. hydrophila from invading EPC (Epithelioma papillosum of carp) cells in vitro. In addition, the proliferative response of head kidney leucocytes of these immune fish was significantly increased as compared to that of the control. The results also showed that the major adhesin could provide significant protective immunity to fish against the challenge by homologous and heterologous strains of A. hydrophila and one virulent strain of Vibrio anguillarum.     

Protection of crucian carp (Carassius auratus Gibelio) against septicaemia caused by Aeromonas hydrophila using specific egg yolk immunoglobulins

Egg yolk immunoglobulins (IgY) were obtained from laying hens immunized with inactivated Aeromonas hydrophila. The purified IgY was shown to inhibit the growth of A. hydrophila in vitro and the optimum concentration for inhibition of A. hydrophila‐specific IgY was 75 mg mL^?1. In a subsequent challenge trial, 100 carp (200~250 g) were assigned to one of ten tanks with ten carp per tank. The fish in one tank were unchallenged whereas the remaining 90 fish were injected intraperitoneally with 100 μL of A. hydrophila at a concentration of 10⁸ cfu mL^?1. For the next 21 days, all fish were moved in their respective groups to a clean tank for 20 min day^?1. The fish in four tanks (one unchallenged tank and three challenged tanks) received no treatment whereas the fish in the remaining six tanks were immersed in either 0.5 g L^?1 aqueous nonspecific IgY (N = 3) or 0.5 g L^?1 aqueous specific IgY (N = 3). Haemoglobin concentrations, white and red blood cell numbers as well as the mortality of specific IgY‐treated fish were significantly different from those of the control. These results suggest that passive immunization by immersion with pathogen‐specific IgY may provide a valuable treatment for A. hydrophila infection in carp.     

Experimental challenge studies in Vietnamese catfish,Pangasianodon hypophthalmus (Sauvage), exposed to Edwardsiella ictaluri and Aeromonas hydrophila

The two main diseases in the pangasius catfish industry are bacillary necrosis of Pangasianodon (BNP) and motile aeromonas septicaemia (MAS), where the aetiological agents have been identified as Edwardsiella ictaluri and Aeromonas hydrophila, respectively. In this study, apparently healthy Pangasianodon hypophthalmus were exposed to E. ictaluri, A. hydrophila or both bacterial species by intraperitoneal injection or immersion. There were 20 fish per treatment group, and the bacterial isolates used for the study were recovered from natural infections of BNP or MAS in farmed Vietnamese P. hypophthalmus. The results of the experimental infections mimicked the natural disease outbreaks reported from these pathogens in P. hypophthalmus. Furthermore, it was clearly demonstrated that E. ictaluri was only recovered from the fish exposed to the bacterium and not recovered from the animals receiving A. hydrophila.     

Investigation on protective effect of recombinant protein (OmpTS) of Aeromonas hydrophila in Common carp (Cyprinus carpio)

The outer membrane protein of Aeromonas hydrophila is a potential candidate for vaccine development. In this study, after cloning and expression of ompTS, 270 common carp, weighing 44 ± 5.7 g divided into five groups, were injected intraperitoneally twice with 3‐week intervals. Groups included the following: PBS, PBS plus Freund's adjuvant, recombinant protein, recombinant protein plus Freund's adjuvant and 20 fish as negative control. Two weeks after the second injection, 30 fish of each group were challenged with a dose of 2 × LD₅₀ of Aeromonas hydrophila and RPS was measured. The antibody level was measured using ELISA test. The protection of recombinant protein in the immunized fish with and without adjuvant, respectively, was about 82.61% and 78.26% (the protection of recombinant protein electroeluted from an SDS–PAGE with and without adjuvant, respectively, was about 78.62% and 69.57%). The average of antibody level in recombinant protein with and without adjuvant was significantly higher than the PBS group (p < .05). The ability of recombinant ompTS to increase the antibody level and to protect the fish from challenge by A. hydrophila demonstrated that recombinant ompTS protein injection can be used to immunize common carp against A. hydrophila infection.     

In vitro inhibition of epithelial cell invasion by Aeromonashydrophila Vibrio species by fish Aeromonas hydrophila major adhesin

A virulent strain of Aeromonas hydrophila (PPD 134/91) was obtained from the Primary Production Department, Singapore. Its major adhesin was isolated and purified by potassium thiocyanate extraction and Bio-Gel P-100 gel filtration. The ability of the protein in peak 1, termed major adhesin, to inhibit bacteria from adhering to and invading host cells was studied in vitro using epithelioma papillosum cells of carp (EPC). Results showed that a concentration of 10 μg ml^–1 of this major adhesin could competitively inhibit 28% of A. hydrophila PPD 134/91 from invading EPC cells in vitro. When the concentration was increased to 40 μg ml^–1, the major adhesin significantly cross-inhibited nine other virulent or weakly virulent strains of A. hydrophila. In addition, the major adhesin significantly inhibited not only another bacterial strain from the same family, Aeromonas sobria, but also strains of Vibrio spp. tested. Therefore, we suggest that the major adhesin of this virulent A. hydrophila strain has the potential to be used as a vaccine against the heterogeneous Aeromonas and Vibrio species.     

Antibacterial efficacy and pharmacokinetic evaluation of sanguinarine in common carp (Cyprinus carpio) following a single intraperitoneal administration

Sanguinarine (SA), with antimicrobial and antiparasitic activities against fish pathogens, exhibits great potential commercial use in aquaculture. However, little information on pharmacokinetics of SA restricts further application in aquaculture. In this study, pharmacokinetics of SA in common carp (Cyprinus carpio) following a single intraperitoneal administration [10 mg kg^?1 BW (body weight)] was evaluated by high‐performance liquid chromatography (HPLC). The peak concentration (C_max) of SA in kidney was 11.8 μg g^?1, which was higher than in other tissues and plasma. The terminal half‐life in fish tissue and plasma was as follows: 42.3 h (kidney) > 37.2 h (liver) > 20.1 h (gill) > 18.8 h (muscle) > 10.9 h (spleen) > 10.0 h (plasma). Additionally, we determined the bacterial loads in tissues of common carp infected with Aeromonas hydrophila after i.p. administration of SA at 0, 5, 10 and 20 mg kg^?1 BW. The results showed that i.p. administration of SA at 10 mg kg^?1 BW significantly enhanced antibacterial efficacy against A. hydrophila, where the antibacterial ratio in the gill, kidney, spleen and liver on day 5 was 95.13%, 93.33%, 90.09% and 92.82%, respectively. Overall, these results suggested the potential of SA to treat A. hydrophila infection in common carp farming industry.     

Improvement of intestinal barrier,immunity, and meat quality in common carp infected by Aeromonas hydrophila using probiotics

The fish pathogen Aeromonas hydrophila causes gastrointestinal tract infections and hemorrhagic septicemia and represents a widespread risk in aquaculture. This study aimed to determine whether compound probiotics could improve the intestinal barrier, immunity, and meat quality of common carp infected by A. hydrophila by feeding them compound probiotics. Carp were assigned to one of four groups: (1) control (no infection, no probiotics); (2) control?+?probiotic; (3) A. hydrophila infected; and (4) the A. hydrophila?+?probiotic group. At the beginning of the experiment, the carp were injected with either saline (0.86%) or A. hydrophila (4.87?×?10⁷ CFU/mL). After 2 weeks of the feeding regime, results suggested that in A. hydrophila infected carp, dietary probiotics regulated the intestinal microflora as evidenced by a reduced abundance of Proteobacteria and increased amounts of Firmicutes and Fusobacteria in the intestine. In addition, dietary probiotics ameliorated both villus swelling and the decrease in villus height induced by A. hydrophila (P?<?0.05). Moreover, probiotics prevented the A. hydrophila-induced decline of Occludin, Claudin-1, and ZO-1 levels in the intestine (P?<?0.05). The addition of probiotics into the feed also increased acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) activity in the serum (P?<?0.05), in comparison to the A. hydrophila group. Importantly, when compared to A. hydrophila-infected carp, compound probiotics alleviated the decrease in muscle nutrient quality (P?<?0.05). In summary, we show that dietary compound probiotics can prevent the impact of A. hydrophila infection on the intestinal function and disease resistance, while ensuring carp muscle quality.

     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

Effects of traditional Chinese medicines (TCM) on the immune response of grass carp (Ctenopharyngodon idellus)

The use of traditional Chinese medicine (TCM) in aquaculture has been widely investigated. It is believed that herbal supplements could enhance the immunity and promote fish growth. The powdered TCM or its decoction can be easily applied in the form of feed supplements by direct incorporation into fish feeds and side effects are seldom found. In this study, Radix scutellaria (Rs) and Rhizoma coptidis (Rc) showed strong bacterial inhibition on Aeromonas hydrophila out of the sixteen selected herbal extracts. Four TCMs, R. scutellaria, R. coptidis, Herba andrographis (Ha), and Radix sophorae flavescentis (Rsf) were selected to form a compound formulation in the ratio of 1:1:2:3. A feeding experiment on grass carp was conducted with addition of the above TCM formulation (0.5, 1 and 2 % w/w) in the fish feed, and bactericidal activity, total protein and immunoglobulin in blood plasma, and nitroblue tetrazolium activity in the whole blood were measured. 2 % supplementation significantly improved (p < 0.05) the bactericidal activity and total immunoglobulin in the plasma after feeding for 21 days. Compared to control, 2 % TCM feeding groups showed a significantly lower mortality after A. hydrophila challenge at the end of experiment and the same result was archived in field trial. In the cost evaluation, the use of the TCM formulation in grass carp culture was insignificant and could be compensated by improved yield. TCM formulation contained different active ingredients including some immuno-stimulant and antimicrobial agents and therefore could be applicable to multiple diseases. Application of this TCM formulation would be a cost-effective and prophylactic approach for disease control in aquaculture, replacing the use of antibiotics for treating enteritis and even other general diseases.     

Indigenous AHL‐degrading bacterium Bacillus firmus sw40 affects virulence of pathogenic Aeromonas hydrophila and disease resistance of gibel carp

Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (10⁹ CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.     

Characterization of a Probiotic Lactobacillus fermentum Isolated from Snakehead,Channa striatus,Stomach

This study sought to isolate and characterize lactic acid bacteria (LAB) from stomach of adult snakehead fish, Channa striatus, to be used as probiotics for freshwater fish. A total of 13 strains were isolated from the stomach of 10 fish, and 4 of these belonged to LAB. Strain LAB‐3 showing highest in vitro growth inhibition of Aeromonas hydrophila in a disk diffusion test was identified as Lactobacillus fermentum by conventional and molecular techniques and evaluated in vitro through various tests. The bacterium could grow at pH 3–8; but the optimum growth was observed at pH 6. Moreover, LAB‐3 grew at 0.15 and 0.3% bile salt concentrations, from 15 to 45 C, and at 4% NaCl. L. fermentum showed in vitro inhibitory activity against three fish pathogens, A. hydrophila, Pseudomonas aeruginosa, and Shewanella putrefaciens, tested by disk diffusion and well diffusion methods. Antibiotic sensitivity tests indicated that L. fermentum was resistant to streptomycin, gentamycin, and kanamycin, intermediate to tetracycline, but sensitive to chloramphenicol, amoxicillin, and ampicillin. Challenge test by using A. hydrophila showed that survival of snakehead was significantly (P < 0.05) improved when 2 × 10⁶ LAB‐3/g was supplemented to the diet. Therefore, this study suggests that L. fermentum might be a promising probiotic in snakehead aquaculture .     

Dietary supplement of Enterococcus faecalis on digestive enzyme activities,short‐chain fatty acid production,immune system response and disease resistance of Javanese carp (Puntius gonionotus,Bleeker 1850)

This study investigated the effects of Enterococcus faecalis on digestive enzyme activities and short‐chain fatty acid production in fish intestine, resistance against Aeromonas hydrophila and humoral immunity response by 3 experiments on Javanes carp (Puntius gonionotus). The experiment 1 revealed that diet supplemented with E. faecalis significantly (P < 0.05) increased protease and lipase activities compared to control fed fish. Moreover, E. faecalis supplementation significantly enhanced the production of propionic and butyric acid in the intestine, while no significant difference (P > 0.05) in acetic acid production was observed. In the challenge study (experiment 2), fish were injected (intraperitoneal) with 10⁷ A. hydrophila per ml and survival was significantly improved when fish were fed diet supplemented with E. faecalis compared to control fish. In experiment 3, dietary E. faecalis affected immune system response as fish fed the probiont and exposed to 10⁶ A. hydrophila per ml displayed significantly elevated antibody levels compared to control fed fish. Fish fed diet supplemented with E. faecalis but not exposed to the pathogen revealed significantly higher antibody level than control fish (P < 0.05). Therefore, E. faecalis can be used as a probiotic in Javanese carp farming.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Lemon,Citrus aurantifolia,peel and Bacillus licheniformis protected common carp,Cyprinus carpio,from Aeromonas hydrophila infection by improving the humoral and skin mucosal immunity,and antioxidative responses

The role of dietary lemon peel (LM) and/or Bacillus licheniformis (BL) on the growth, immunity, and resistance against Aeromonas hydrophila in common carp, Cyprinus carpio was investigated in this study. LM and BL were included in diets at 0% (T0), 10⁸ CFU/g BL (T1), 1.5% LM and 10⁸ CFU/g BL (T2), and 3% LM and 10⁸ CFU/g BL (T3). Fish fed with T1, T2, or T3 had higher weight gain, specific growth rate, white blood cells count, and blood total protein with lower feed conversion ratio than T0 group (p < .05). The albumin increased significantly (p < .05) in fish fed both BL and LM (T3). The serum superoxide dismutase (SOD), catalase, lysozyme, and bactericidal activities were significantly increased in fish fed both BL and LM (T2 and T3), while serum glutathione peroxidase increased in fish fed BL (T2) (p < .05). Fish fed T1, T2, and T3 diets displayed higher SOD and lower malondialdehyde than fish fed T0 (p < .05). After the A. hydrophila challenge, the mortality rate was significantly lower in T1, T2, and T3 groups than the T0 group (p < .05). The obtained results revealed that LM and BL could be used to increase resistance against A. hydrophila infection in carp. However, further field studies should be performed to confirm the obtained results.     

Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti‐A. hydrophila and anti‐V. fluvialis formalin‐killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non‐optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.     

Effects of dietary supplementation with probiotic live yeast Debaryomyces hansenii on the immune and antioxidant systems of leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila

Two trials were conducted to determine the efficacy of fish fed live yeast Debaryomyces hansenii strain CBS 8339 on immune and antioxidant systems in leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila. Juveniles (12±0.5 g) were fed with a control diet or a D. hansenii‐supplemented diet (10⁶ colony‐forming units per gram) for 5 weeks. The live weight of fish was registered on a weekly basis. After 4 weeks, fish from each treatment were immunocompromised with pathogenic A. hydrophila and further fed for 1 week in order to evaluate the effect on immunological and antioxidant parameters. Generally, the results showed enhanced growth performance in fish fed the diet containing yeast compared with the control. Addition of live yeast had no significant effect on the immunological parameters after 4 weeks of feeding. However, post infection with A. hydrophila fish fed the yeast‐supplemented diet resulted in a significant increase in the levels of plasmatic immunoglobulin M. Superoxide dismutase and catalase (CAT) activities were significantly higher in the yeast group. In this fish, CAT and heat shock protein 70 genes were up‐regulated before and after infection of A. hydrophila. The present study is the first one reporting that yeast (D. hansenii) can enhance immunity and resistance against A. hydrophila.     

Enhancement of the innate immune system and disease‐resistant activity in Cyprinus carpio by oral administration of β‐glucan and whole cell yeast

The effects of dietary β‐ (1,3) glucan and whole cell yeast (Sacharomyces uvarum) on the immune response and disease resistance to Aeromonas hydrophila were investigated in Cyprinus carpio. β‐(1,3) glucan was extracted from the yeast. Both β‐(1,3) glucan and whole yeast were incorporated into the diet at 1% level and fed to common carp C. carpio for a period of 60 days. Control and treated fish were exposed to A. hydrophila on the 30th and the 60th day of the experimental period. Dietary supplementation of glucan significantly increased the white blood cell count in fish on the 60th day (2.91±0.04 × 10⁴), and the highest nuetrophil nitro blue tetrazolium (NBT) activity was also observed in glucan‐fed fish (30th day). A consistent increase in neutrophil (NBT) activity was also observed in whole cell fed fish until the end of the experiment. Similarly, β‐(1,3) glucan and whole cell yeast enhanced the serum lysozyme activity from the 15th day onwards but higher activity was reported on the 30th day in glucan and the 60th day in whole cell yeast‐fed fish. Suplementation of β‐(1,3) glucan protected the fish from A. hydrophila infection. Nearly 75–80% of the fish survived pathogen exposure (relative percentage survival). However, only 54–60% survival was observed in the whole cell‐fed fish. β‐(1,3) glucan and whole cell yeast protect the fish from pathogens by enhancing the cellular and humoral immune response in C. carpio.     

Effect of dietary willow herb,Epilobium hirsutum extract on growth performance,body composition,haematological parameters and Aeromonas hydrophila challenge on common carp,Cyprinus carpio

The present study was conducted to evaluate the use of Epilobium hirsutum extract in diet of common carp, Cyprinus carpio (20 ± 2 g). Different levels of plant extract (0%, 0.5%, 1, 3% and 3%+2‰ multivitamin (2 g of multivitamin per kg diet) were spread on commercial diet. The feed was offered by 8 weeks. Results showed that fish fed experimental diets had no significant difference (P>0.05) in specific growth rates, condition factor, feed conversion ratios and survival compared with control. There were no significant difference (P>0.05) in moisture, crude protein, crude lipid and ash content of common carp fed diets containing various levels of plant extract. The mortality of fish challenged by Aeromonas hydrophila have been recorded for 30 days after challenging, results showed that mortality decreased significantly (P<0.05) in fish fed diet containing plant extract compared with the control. The lowest mortality observed in group fed diet containing 3% plant extract. Haematological parameters showed that white blood cells had significantly (P<0.05) increased in infected and uninfected groups compared with the control. Red blood cells, haemoglobin and haematocrit had no significant change (P>0.05) in infected and uninfected groups compared with the control.     

Effects of dietary pyridoxine on disease resistance,immune responses and intestinal microflora in juvenile Jian carp (Cyprinus carpio var. Jian)

This experiment was conducted to evaluate the effects of dietary pyridoxine on disease resistance, immune responses and intestinal microflora of fish. A total of 1050 Jian carp (11.71 ± 0.05 g) were randomly distributed into seven groups, feeding diets containing graded levels of pyridoxine (0.2, 1.7, 3.2, 5.0, 6.3, 8.6 and 12.4 mg kg^?1 diet). After 80 days of feeding, a challenge trial was conducted by injection of Aeromonas hydrophila for 17 days. Results indicated that with increasing dietary pyridoxine concentration up to 5.0 mg kg^?1 diet, survival rate after challenge with A.hydrophila and phagocytic activity of leukocyte were improved (P < 0.05), and plateaued thereafter (P > 0.05). Red blood cell and white blood cell counts were lowest when fed the diet containing 1.7 mg pyridoxine kg^?1 diet. Haemagglutination titre, lysozyme activity, acid phosphatase activity, total iron‐binding capacity, antibody titre and immunoglobulin M content followed the similar pattern to that observed with survival rate. Aeromonas hydrophila, Escherichia coli and Lactobacillus counts in intestine were not affected by dietary pyridoxine concentration (P > 0.05). These results suggested that pyridoxine could enhance immune response of fish.