EGCG: Potential application as a protective agent against grass carp reovirus in aquaculture

Grass carp reovirus (GCRV) is the primary cause of grass carp haemorrhagic disease. The major catechin in green tea, (–)‐epigallocatechin‐3‐gallate (EGCG), has been found to have anti‐GCRV activity in the C. idellus kidney cell line (CIK). The aim of this study was to test the potential application of EGCG as an anti‐GCRV agent in aquaculture. Here, we demonstrate that various concentrations (99%, 50% and 35%) of EGCG could inhibit GCRV infectivity. EGCG (50%) + GCRV treatment significantly reduced the number of dead fish at 1‐, 2‐, 3‐, 4 ‐and 5‐day post‐challenge compared with the negative control (GCRV challenge without EGCG treatment). The safety of EGCG compound products on cell survival was studied using four fish cell lines; we did not detect a significant change in cell viability within 24 hours of EGCG incubation. We also evaluated toxicity and concentrations of malondialdehyde (MDA), glutathione (GSH) and lysozyme (LZM) in the grass carp, and the results showed that even a high dose of EGCG did not induce toxicity. Following EGCG compound injection, the concentration of MDA decreased and the concentration of GSH and LZM increased compared with the control groups. We also detected EGCG concentration in grass carp plasma and kidney using HPLC with electrochemical detection after intraperitoneal injection at a dose of 150 mg/kg. The concentration of EGCG in the plasma and kidney reached the highest levels (20 μg/ml and 1.5 μg/ml) about 12 hr after injection and then decreased. Overall, EGCG is a safe, effective product that could inhibit GCRV infection and improve immunoactivity in aquaculture.     

Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single‐stranded DNA (ssDNA) aptamers were selected against GCRV‐infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem‐loop structures, and GVI‐11 had the lowest ΔG value of ?30.84 KJ/mol. Three aptamers could specifically recognize GCRV‐infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66 nM for aptamers GVI‐1, GVI‐7 and GVI‐11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI‐1, GVI‐7 and GVI‐11 on the surface of GCRV‐infected cells could be membrane proteins, which were trypsin‐sensitive. Furthermore, FAM‐labelled aptamer GVI‐7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV‐infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture.     

Pharmacokinetics and tissue distribution of bronopol in grass carp,Ctenopharyngodon Idella,at 15 and 20°C

This study explored the pharmacokinetic characteristics of bronopol in grass carp (Ctenopharyngodon idella) under different temperatures. The concentrations of bronopol in the plasma and tissues (kidney, liver and muscle) of grass carp after soaking in bronopol (6.75 µg/ml) at 15 and 20°C were determined using liquid chromatography–mass spectrometry. The concentrations of bronopol in both plasma and tissue first increased and then decreased with soaking time at both tested temperatures. The peak bronopol concentrations at 15°C (plasma: 8.934 µg/ml; kidney: 9.23 µg/g; liver: 9.47 µg/g; and muscle: 7.98 µg/g) were slightly lower than those at 20°C (plasma: 9.654 µg/ml; kidney: 10.83 µg/g; liver: 13.40 µg/g; and muscle: 8.68 µg/g). In an analysis of pharmacokinetic parameters, the bronopol concentration–time profiles for plasma were best described by a two‐compartment, open pharmacokinetic model with first‐order absorption. The t_1/2β for plasma at 15 and 20°C was 82.933 and 92.041 hr, respectively, indicating that the elimination of bronopol was faster at 15°C than at 20°C. In addition, AUC_0‐t and AUC_0‐∞ values of bronopol were 477.892 and 495.809 µg/L hr at 15°C and 495.809 and 589.859 µg/L hr at 20°C, indicating that the content of bronopol in the organism was higher at higher temperature.     

In vivo antiviral efficacy of moroxydine hydrochloride against grass carp reovirus and the drug safety assessment

Our previous studies have verified that moroxydine hydrochloride (Mor) could inhibit replication of grass carp reovirus (GCRV) and suppress apoptosis of Ctenopharyngodon idella kidney cells, but be lack of information whether exists on antiviral activity in vivo. The paper was undertaken to explore the antiviral response of Mor against GCRV in grass carp and investigate the safety of drug for aquatic organisms. The results showed that injection treatment of Mor could more effectively inhibit GCRV replication than immersion administration. All the RNA systheses of vp3 and vp6 on day 7 in head kidney, gill, hepatopancreas and dorsal muscle in the Mor injection group were lesser than 0.07‐fold than that of in control group. And the GCRV‐inducing grass carp mortality was effectively controlled within 7 days post Mor injection therapy. Additionally, the reduction of superoxide dismutase activity, total antioxidant capacity and catalase activity in serum was effectively controlled by Mor. Moreover, drug safety assessment results showed that 500 mg/L of Mor was safe to C. idella, Bacillus subtilis, Chlorella vulgaris and Tetrahymena thermophila, which was far higher than the therapeutic concentration. The present study proved Mor as harmless formulations or products had potential value in the control of GCRV in aquaculture, with the advantage of super in vivo antiviral activity and environmental safety.     

草鱼呼肠孤病毒VP6蛋白基因植物表达载体的构建

以据草鱼呼肠孤病毒(GCRV)VP6蛋白的全基因序列(GeneBank AF403394)为模板,采用RT-PCR构建了草鱼呼肠孤病毒VP6蛋白基因植物表达载体的构建。结果显示:实验构建的pCR 2.1-VP6重组质粒含有NcoⅠ和BglⅡ酶切位点;pCR 2.1-VP6重组质粒经PCR扩增和测序显示含有1.3 Kbp的草鱼呼肠孤病毒VP6基因读码框片段。将目的片段VP6酶切、克隆到携带有绿色荧光蛋白(GFP)基因的植物表达载体pCAMBIA1302中,再经酶切、PCR扩增和序列测定显示,pCAMBIA1302-VP6含有1.3 Kbp的草鱼呼肠孤病毒VP6基因片段,说明已插入植物表达载体pCAMBIA1302绿色荧光蛋白基因前,成功构建了融合表达草鱼呼肠孤病毒VP6蛋白和绿色荧光蛋白的植物表达载体pCAMBIA1302-VP6。     

草鱼病毒性出血病研究进展

草鱼出血病是严重困扰着草鱼养殖业发展的一大疾病。根据近几十年来对于该病研究的进展,就草鱼出血病病原的生物学特性、不同发现株之间的比较、分子生物学研究以及疾病的防治等方面进行综合评述,总结了草鱼出血病的研究现状及研究中亟待解决的问题,并提出了该领域以后的重点研究方向。为进一步深入开展对该病的防治研究提供参考,以提高草鱼养殖的生产率。     

Protein‐sparing capability of dietary lipid in herbivorous and omnivorous freshwater finfish: a comparative case study on grass carp (Ctenopharyngodon idella) and tilapia (Oreochromis niloticus × O. aureus)

Two, 8‐week feeding trials were conducted to compare protein‐sparing capability of dietary lipid in herbivorous grass carp (Ctenopharyngodon idella) and omnivorous tilapia (Oreochomis niloticus × O. aureus). Utilizing a 2 × 3 factorial design, experimental diets containing two levels of crude protein (380 and 250 g kg⁻¹) and three levels of lipid (0, 40 and 100 g kg⁻¹) were formulated for use in both feeding trials. Growth performances showed better response of both fish fed 380 g kg⁻¹ protein diet than those fed 250 g kg⁻¹ protein diet. Despite the dietary protein level, weight gain (WG), specific growth ratio (SGR), feed conversion ratio (FCR) and protein efficiency ratio were much higher (P < 0.05) for grass carp fed 40 g kg⁻¹ lipid diet than those fed 100 g kg⁻¹ lipid diet; however, there were no significant differences in tilapia fed the two diets. The feed intake of grass carp fed lipid‐free diet was the lowest, but it tended to decrease with increase in dietary lipids in tilapia. Lipid retention (LR) was negatively correlated with dietary lipid concentration of both fish. Viscerosomatic index (VSI), hepatosomatic index (HSI), intraperitoneal fat ratio (IPF) and whole‐body and liver lipid content positively correlated with dietary lipid concentration of both fish. Plasma parameters and liver enzymes activities were also positively correlated with dietary lipid concentration of both fish. Liver lipid contents were higher and enzymes activities were lower in grass carp when compared with tilapia. These data suggested that there was no evidence of a protein‐sparing effect of dietary lipids in grass carp. Tilapia has relatively higher capacity to endure high dietary lipid level compared to grass carp.     

Dietary nano‐selenium enhances antioxidant capacity and hypoxia tolerance of grass carp Ctenopharyngodon idella fed with high‐fat diet

To evaluate the effects of dietary nano‐selenium (Nano‐Se) on antioxidant capacity and hypoxia tolerance of grass carp fed with high‐fat diet, experimental fishes were fed Nano‐Se supplemented diets at doses of 0 (Control), 0.3, 0.6, 0.9 and 1.2 mg/kg for 10 weeks. After feeding trial, a part of the fishes were exposed to hypoxia stress. Results showed that the survival ratio of grass carp significantly increased in 0.6 and 0.9 mg/kg Nano‐Se group, and the content of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) significantly decreased in 0.6–1.2 mg/kg Nano‐Se groups compared with the control group. In addition, dietary Nano‐Se significantly enhanced glutathione peroxidase (GPX) activity and reduced the malondialdehyde (MDA) content in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. Dietary Nano‐Se significantly elevated mRNA expression of GPX1 and catalase (CAT) by promoting the mRNA expression of NF‐E2‐related nuclear factor 2 (Nrf2) in the hepatopancreas. After hypoxia stress, the GPX and superoxide dismutase (SOD) activities were significantly enhanced, and the MDA content and mortality rate consequently decreased in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. In summary, these results suggested that optimal Nano‐Se in diet enhanced the antioxidant capacity and hypoxia tolerance of grass carp.     

Growth performance,digestive and absorptive capacity of on‐growing grass carp (Ctenopharyngodon idellus) fed with graded level of dietary fibre from soybean hulls

The present research evaluated the effects of graded levels of dietary fibre on growth, digestive and absorptive capacities, and the potential underlying mechanisms in on‐growing grass carp (Ctenopharyngodon idella). Grass carp (123.00 ± 0.70 g) were fed diets with graded levels of neutral detergent fibre (NDF: 97.8, 119.0, 140.2, 159.4, 181.2 and 201.6 g/kg diet) for 60 days. Besides, a 2‐week digestion experiment was conducted to explore the effect of dietary fibre on the apparent digestibility coefficients (ADC) of feed. The results showed that optimum dietary fibre level (140.2 or 159.4 g/kg diet) increased feed intake, per cent weight gain, specific growth rate and the ADC of dietary protein (p < .05); increased the activities of digestive and brush border enzyme; and up‐regulated intestinal amino acid transporter mRNA levels (SLC1A5 and SLC6A19b, p < .05) partially associated with activation of TOR signalling pathway. However, high dietary fibre levels (NDF levels ≥181.2 g/kg diet) were not conducive to the digestion and absorption of nutrients, resulting in the decline of growth performance. Thus, based on the quadratic regression analysis for per cent weight gain and feed efficiency, the optimum (143.9 and 134.5 g/kg diet) and maximum tolerance (189.8 and 171.2 g/kg diet) levels of dietary NDF were estimated for on‐growing grass carp.     

Comparison study of three compounds in Eucommia ulmoides on growth,flesh quality of grass carp (Ctenopharyngodon idella)

This study was conducted to compare the growth‐promoting and flesh quality ‐improving effects of three active compounds in Eucommia ulmoides (EU) on grass carp (Ctenopharyngodon idella). Four iso‐nitrogenous diets supplemented with 400 mg/kg inclusion of geniposidic acid (GA), chlorogenic acid (CGA), geniposide (GP) and their combination (GA:CGA:GP = 1:1:1, the mixture) were prepared and fed to grass carp (47.1 ± 0.6 g) for 75 days. The results indicated that weight gain was increased by 5.22%, and feed conversion ratio decreased by 0.07 by dietary CGA (p < 0.05). In flesh quality, the four supplementations significantly increased muscle fibre density, total collagen and alkaline‐insoluble collagen in skin, and reduced steaming loss of flesh. In addition, dietary CGA, GP and the active compounds mixture further increased total collagen, alkaline‐insoluble collagen and amino acid in flesh. In collagen genes expression, the expression of COL1A1 in muscle and skin was significantly promoted by the supplementation of GA, CGA, GP and their combination (p < 0.05). In conclusion, the supplementation of GA, CGA, GP and their combination improved the flesh quality of grass carp, and the growth was increased by CGA. CGA played more important roles in growth‐promoting and flesh quality‐improving effects than GP and GA.     

草鱼成纤维细胞gcngr1基因的表达分析

Ngr1(nogo-66 receptor)是在哺乳类上发现的一种神经元受体,可调节轴突的可塑性并抑制损伤中枢神经系统(CNS)的再生,最新研究还发现它是哺乳动物呼肠孤病毒(mammalian reovirus)的神经受体。草鱼呼肠孤病毒(grass carp reovirus,GCRV)可引发草鱼(Ctenopharyngodon idellus)出血病导致高死亡率,开展GCRV受体相关研究可有助于了解病毒的致病机制。该研究在草鱼吻端成纤维细胞(PSF)中克隆到草鱼ngr1 c DNA序列(下文简称为gcngr1),发现其与哺乳动物呼肠孤病毒神经受体基因Ngr1有相似的结构序列;采用q RT-PCR方法检测该基因在PSF细胞的表达情况,结果显示受草鱼呼肠孤病毒(GCRV-GD108株)感染后gcngr1 m RNA的表达量显著上升,与病毒的增殖趋势基本一致;病毒经病毒抗体孵育后再感染PSF细胞,细胞中病毒的增殖水平下降,gcngr1m RNA的表达量也显著下降。该研究结果提示gcngr1与病毒的感染相关,为进一步分析gcngr1是否为GCRV神经元受体提供依据。     

Effects of dietary chlorogenic acid on growth,flesh quality and serum biochemical indices of grass carp (Ctenopharyngodon idella)

The study was to investigate effects of dietary chlorogenic acid (CGA) on growth performance, flesh quality and serum biochemical indices of grass carp (95.1 ± 0.3 g) (Ctenopharyngodon idella) fed seven different diets, including control diet, Eucommia ulmoides (EU)‐supplemented diet (20 g kg^–1) and CGA‐supplemented diets containing 100, 200, 400, 600 and 800 mg/kg CGA. Contents of collagen and alkaline‐insoluble collagen in muscle and skin were significantly increased by dietary CGA and EU (p < .05). Total essential amino acids (TEAA) and total amino acids (TAA) in muscle of grass carp fed EU diet or 400, 600 and 800 mg/kg CGA diet were significantly higher than those of fish fed control diet and 100 and 200 mg/kg CGA diet (p < .05). Fish fed 200–800 mg/kg CGA showed significantly lower muscle crude lipid content than EU, control and 100 mg/kg CGA groups (p < .05). Fish fed CGA‐supplemented diets (100–800 mg/kg) had significantly higher muscle fibre density and lower muscle fibre diameter than control group (p < .05). In conclusion, supplementation of CGA improved flesh quality of grass carp, and supplemental level of CGA for improving flesh quality and growth was estimated to be 400 mg/kg diet.     

Dietary α‐Ketoglutarate supplementation alleviates harmful effects of high environmental ammonia on grass carp,Ctenopharyngodon idella

Current intensive fish farming usually causes high environmental ammonia (HEA) in ponds that is toxic to fish. α‐Ketoglutarate (α‐KG) can be rapidly transaminated to glutamic acid and further aminated to glutamine. Therefore, we hypothesized that dietary α‐KG supplementation would alleviate HEA toxicity to fish. To test the hypothesis, 270 healthy grass carp (Ctenopharyngodon idella) juveniles were randomly assigned to control, HEA (18.37 mg/L ammonia) and HEA + α‐KG (0.75% of α‐KG) groups. Ammonia and free amino acid content in plasma and brain, liver glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activity, and urea and glycogen content were measured on the first, seventh and 42nd days. Our results showed short‐term HEA exposure (1 day) led to a significant ammonia accumulation in the brain and plasma and significantly decreased glutamic and aspartic acid content in the brain and increased glutamine content in the brain and plasma. The long‐term HEA exposure (42 days) caused significant reductions in glycine and arginine content in the brain tissue. In most cases, dietary α‐KG supplementation alleviated the fluctuations in FAA content in the brain and plasma. Our results suggested dietary α‐KG alleviated HEA toxicity to grass carp.     

Dietary Moringa oleferia leaf meal induce growth,innate immunity and cytokine expression in grass carp,Ctenopharyngodon idella

The present research was designed to investigate the growth promoting and immunostimulating properties of Moringa oleferia leaf meal (MLM) in grass carp. Juvenile grass carp (22.03 g ± 1.164) were fed with diets supplemented with 0, 50, 100 and 150 g/kg MLM for 48 days. At the end of feeding trial, skin mucus was used for analysis of lysozyme, protease, antiprotease and peroxidase activity. Head kidney was used for expression analysis of tumour necrosis factor‐alpha, interleukin‐8 and interferon‐γ. The obtained results showed that fish fed with 100 and 150 g/kg MLM had significant increase in weight gain and specific growth rate (p < .05). However, condition factor was not altered. The MLM (50 and 100 g/kg) inclusion resulted in higher mucus lysozyme and protease activity (p < .05), while peroxidase activity increased only in fish fed with 100 g/kg MLM and antiprotease activity was not altered. Expression of tnf‐α increased in a dose‐dependent manner, and significant (p < .05) increase was recorded in fish fed with 150 g/kg MLM. The expression of il‐8 and ifn‐γ increased in fish fed with 50 and 150 g/kg MLM; however, the increase was not significant (p > .05). In conclusion, supplementing juvenile grass carp feed with MLM up to 150 g/kg has growth promoting and immunostimulating effects.     

Two biomass preparation methods provide insights into studying microbial communities of intestinal mucosa in grass carp (Ctenopharyngodon idellus)

Animal digestive tract is habitat for a large number of autochthonous microbiota, which play central roles in multiple biological and physiological processes of the host. In this study, two different micro‐biomass preparation methods were employed to evaluate the diversity of intestinal mucosa‐associated microbiota in grass carp (Ctenopharyngodon idellus). Genomic DNAs were isolated either directly from intestinal mucosal samples (group A), or from micro‐biomass after microbial dissociation (group B). Community richness, diversity and evenness indices were all higher in group B, but differences were not statistically significant (P = 0.97, P = 0.33, P = 0.34 respectively). Furthermore, group B samples exhibited an increased ratio of bacterial DNA in comparison with group A samples, but the difference was also not statistically significant (P = 0.74). In addition, there were no statistically significant differences between the two groups (P > 0.05) at the taxonomic level. Our results support previous findings that there exists a great abundance of the intestinal mucosa‐adherent microbiota in the grass carp; among these, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Spirochaetes and Fusobacteria were the most common phyla. Within these microbiota, Paenibacillus, Bacteroides, Bacillus and Cetobacterium genera comprise the majority of the community, implicating their functional importance (e.g. as probiotics) to their host. Our results contribute towards a better understanding of the intestinal microbial profile of grass carp. Both micro‐biomass preparation techniques proved to be feasible for studying mucosa‐adherent microbiota of grass carp; however, the second method (group B) provides a protocol that is somewhat more effective than the first method (group A).     

草鱼呼肠孤病毒JX-0902株的分离和鉴定

收集江西南昌地区患典型草鱼出血病的病鱼组织,进行超薄切片电镜观察,结果显示,在脾、肾样品中发现大量病毒颗粒,病毒无囊膜,近球形,直径约70 nm,形态与已报道的草鱼呼肠孤病毒(grass carp reovirus,GCRV)相似。取病鱼肌肉、内脏研磨,组织液经离心、过滤除菌后进行鱼体人工感染试验和细胞感染实验,结果发现,人工感染试验鱼出现死亡,且具有典型出血症状;组织滤液感染草鱼肾细胞系(CIK)细胞后,盲传6代未观察到典型细胞病变效应,但感染细胞固定后经超薄切片电镜观察,细胞质内有大量病毒存在,与病鱼组织切片观察到的病毒形态一致。SDS-PAGE结果进一步揭示,新分离病毒株(暂命名JX-0902)基因组由11条dsRNA组成,呈现水生呼肠孤病毒基因组典型特征,但基因组带型与GCRV 873株存在差异,而与HZ08株接近。根据GenBank上已提交的草鱼呼肠孤病毒S6序列(GQ896337)设计特异引物,以JX-0902株的cDNA作为模板,可扩增出特异性条带。基于S6序列的聚类分析结果显示,JX-0902与GCRV HZ08株、GD108株S6同源性高达98%、99%,该分离株应为草鱼呼肠孤病毒。     

Depuration and starvation improves flesh quality of grass carp (Ctenopharyngodon idella)

Farmed grass carp (Ctenopharyngodon idella) at commercial size were transported to a natural lake for long‐term depuration while being food deprived. The effect of depuration time on the quality of fish fillets was investigated based on proximate compositions, textural parameters and flavour characteristics. The results showed that protein and lipids, but not carbohydrates, were the major source of energy for grass carp during depuration and starvation. Textural parameters that included hardness, springiness, gumminess and cohesiveness increased significantly after depuration, as well as water‐holding capacity of fish muscle. Taste and odour characteristics of grass carp muscle were obviously changed by depuration based on tests by an electronic tongue and nose. Off‐flavour volatile compounds, such as nonanal and hexanal, were reduced after depuration. In conclusion, the quality of grass carp fillets was improved effectively by long‐term depuration and food deprivation. More than 20 days of depuration was appropriate for the enhancement of grass carp quality before marketing.     

Growth performance,intestinal morphology,hepatopancreatic antioxidant capacity and growth‐related gene mRNA expressions of juvenile grass carp (Ctenopharyngodon idellus) as affected by graded levels of dietary arginine

An 8‐week growth trial was conducted to investigate the effects of dietary Arg levels (7.6, 12.3, 17.9, 22.4 and 28.6 g/kg diet) on growth performance, hepatopancreatic antioxidant capacity, intestinal morphology and growth‐related gene expressions of juvenile grass carp (Ctenopharyngodon idellus). The results showed that SGR in Arg22 and Arg28 groups was lower than in Arg12 and Arg17 groups (p < 0.05). Serum NO content in Arg7 group was lower than other groups. Hepatopancreatic GSH‐Px activity was higher in Arg17 group than in Arg7 group, while MDA content showed the opposite trend. Hepatopancreatic IGF‐1 expression tended to increase with Arg from 7.6 to 22.4 g/kg and then decreased in Arg28 group (p < 0.05), while IGFBP‐1 expression increased with Arg level. Muscle mRNA expressions of TOR and S6K1 showed quadratic trends as dietary Arg level increased, which were higher in Arg17 group than in Arg22 and Arg28 groups (p < 0.05). Higher mRNA expression levels of y⁺LAT1, y⁺LAT2 and PepT1, as well as higher villus height and villus width in foregut, were all observed in Arg17 group. The optimal dietary Arg level based on SGR by the quadratic model was 15.3 g/kg diet for juvenile grass carp, corresponding to 54.7 g/kg dietary protein.     

Identification of immunogenic proteins of Flavobacterium columnare by two‐dimensional electrophoresis immunoblotting with antibacterial sera from grass carp,Ctenopharyngodon idella (Valenciennes)

Flavobacterium columnare is a Gram‐negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F. columnare using an immunoblotting approach in two‐dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti‐grass carp‐recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF/TOF MS). The 14 proteins are immunogenic molecules of F. columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl‐CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose‐bisphosphate aldolase, 3‐hydroxybutyryl‐CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.