Glucan administration potentiates immune defence mechanisms of channel catfish, Ictalurus punctatus Rafinesque

Abstract. Intraperitoncal injection of β-l,3 glucan in channel catfish, Ictalurus punctatus Rafinesque, greatly reduces mortality from experimental infection with Edwardsiella ictaluri. Anterior kidney phagocytes from fish receiving glucan had enhanced phagocytic and bactericidal ability. The elevated bactericidal ability of phagocytes was not accompanied by increased production of hydrogen peroxide. Fish injected with glucan responded to subsequent E. ictaluri immunizations with higher serum antibody titres relative to the control catfish. The timing of glucan administration and antigen immunization was also important. These results indicate that β-1,3 glucan potentially could be utilized prophylactically as an immunomodulator in channel catfish.     

Use of an indirect enzyme-linked immunosorbent assay (elisa) in the detection of channel catfish, Ictalurus punctatus (Rafinesque), antibodies to Edwardsiella ictaluri

Abstract. The use of an indirect enzyme-linked immunosorbent assay ( elisa ) for the detection of channel catfish antibody to Edwardsiella ictaluri is described. Changes in agglutination titre in fish immunized with Edwardsiella ictaluri heat killed whole bacterins or lipopolysaccharides were reflected by corresponding changes in elisa readings. Relatively high correlations were observed among elisa OD readings, computed elisa titres and corresponding agglutination titres.     

Edwardsiella ictaluri bacteraemia elicits shedding of Aeromonas hydrophila complex in latently infected channel catfish, Ictalurus punctatus (Rafinesque)

Edwardsiella ictaluri is a primary bacterial pathogen of channel catfish, Ictalurus punctatus, and the causative agent of enteric septicaemia of catfish . Edwardsiella ictaluri is known to gain entry to the host by infection of the nares, gastrointestinal tract, and gills, and to disseminate to organs via an as yet uncharacterized acute bacteraemia. In this study, fluorescent microscopy showed E. ictaluri on the gill within 5 min of immersion challenge and E. ictaluri could also be isolated from the circulation within 5 min. When removed to clean water, catfish cleared circulating bacteria within 15 min and the blood remained free of E. ictaluri until its reappearance at the 12 h post-infection sampling. However, Aeromonas hydrophila , the aetiological agent of motile aeromonad septicaemia, appeared within the circulation 7 h post-challenge with E. ictaluri and was detected in all fish at 12 h post-infection. Only 20% of fish carried A. hydrophila in the trunk kidney that could be detected by plate culture on Rimler–Shotts agar; however, 100% of challenged and stress-control fish were A. hydrophila complex positive at 24 h post-challenge. These results suggest that although the catfish is capable of clearing its circulation of E. ictaluri , superinfection with latent A. hydrophila may enhance clinical signs of edwardsiellosis. This is the first report of a bacterial superinfection appearing in fish.     

An assessment of the antigenic homogeneity of Edwardsiella ictaluri using monoclonal antibody

Abstract. Monoclonal antibodies were made against the reference strain of Edwardsiella ictaluri (ATCC 3320). Antibody produced by one of seven anti- E. ictaluri hybridomas reacted positively by the immunofluorescent antibody technique against 17 other E. ictaluri isolates. All hybridoma antibodies failed to react with six other bacterial species pathogenic to fish including E. tarda . Ouchterlony tests indicated that four anti- E. ictaluri clones produced only one kind of immunoglobulin. Electrophoresis of 14 different E. ictaluri isolates indicated identical protein bands at 36 and 60 kilodaltons (KD) in all isolates except an isolate from Thailand. Using the immunoblot method, channel catfish anti- E. ictaluri serum reacted with protein bands at 34 and 60 KD, which indicates that this molecular weight protein in the bacterium may be the dominant immunoprotein.     

Susceptibility of five species of fish to Edwardsiella ictaluri

Abstract. Five species of fingerling fish, channel catfish, Ictalurus punctatus , tilapia, Sarotherodon aureus , golden shiner, Notemigonus crysoleucas , largemouth bass, Micropterus salmoides and bighead carp, Aristichthys nobilis , were tested to determine their susceptibility to the bacterium, Edwardsiella ictaluri , at 26°C, Channel catfish demonstrated high susceptibility to E. ictaluri as 100% of those fish injected with 1.5 × 10³ cells died within 10 days. Tilapia demonstrated slight susceptibility to the pathogen while golden shiner, bighead carp and largemouth bass were not susceptible. E. ictaluri was isolated from a higher percentage of peritoneal cavities, livers and kidneys of channel catfish than of other species. Sequential growth of E. ictaluri in the liver of channel catfish is described.     

Serological detection of Edwardsiella ictaluri Hawke lipopolysaccharide antibody in serum of channel catfish, Ictalurus punctatus Rafinesque

Abstract. Bacterial agglutination, passive haemagglutination, complement-dependent passive haemolysis, indirect immunofluorescence, agar gel immunodiffusion and agglutination with fractions of immunized fish serum were compared for detecting humoral antibody to the lipopolysacchande (LPS) of Edwardsiella icialuri Hawke in channel catfish. Bacterial agglutination titres averaged 1: 672; passive haemagglutination titres averaged 1: 1152; and complement-dependent haemolysis titres averaged 1: 2360. Serum from non-vaccinated fish ranged from 0 to 1:32. Indirect fluorescence and immunodiffusion demonstrated positive reactions to the LPS antibody. Fractionation of immune sera produced three fractions, one of which strongly haemagglutinated E. ictaluri but the other two did not. All six serological techniques were sensitive to E. ictaluri LPS antibody.     

鲖爱德华菌口服疫苗对斑点鲖的免疫效果

为评价鲖爱德华菌口服微球疫苗对斑点鲖的免疫效果,实验以天然高分子聚合物海藻酸钠和鲖爱德华菌灭活疫苗为材料,制备鲖爱德华菌口服微球疫苗。将实验动物随机分为鲖爱德华菌微球疫苗组、鲖爱德华菌灭活疫苗组、空微球组和对照组,以拌料口服方式进行免疫,通过检测血清中溶菌酶活力、总超氧化物歧化酶(T-SOD)活力、补体替代途径(ACH50)活性等非特异性免疫指标,抗体效价以及相对免疫保护率评价疫苗免疫效果,采用荧光定量PCR检测口服疫苗对斑点鲖免疫相关基因表达量的影响。结果显示,鲖爱德华菌口服微球疫苗能够较长时间增强斑点鲖非特异性免疫功能;血清凝集效价于第5周达到峰值,为1∶16,免疫后第7周仍可检测到特异性抗体;口服鲖爱德华菌微球疫苗的斑点鲖获得的抗鲖爱德华菌相对免疫保护率为60.7%,远高于灭活疫苗组(14.3%)及空微球组(10.7%);荧光定量分析结果显示,攻毒后48 h相比攻毒前各免疫基因表达量均有上调,鲖爱德华菌微球疫苗对受免鱼肾脏、脾脏中免疫基因的表达影响尤为明显。结果表明,鲖爱德华菌口服微球疫苗能增强斑点鲖非特异性免疫功能,对鲖爱德华菌病起到一定的预防作用。     

Uptake and Clearance of Edwardsiella ictaluri in the Peripheral Blood of Channel Catfish Ictalurus punctatus Fingerlings during Immersion Challenge

Uptake and clearance of Edwardsiella ictaluri in the peripheral blood of channel catfish Ictalurus punctatus fingerlings were monitored for 216 h after exposure to E. ictaluri for 4 h and 8 h under static conditions. Most fish exposed to E. ictaluri developed bacteriemia 24 h post-exposure, and by 72 h post-exposure E. ictaluri was recovered from all the blood of all exposed fish. The number of E. ictaluri colony forming units (CFU) in the blood of moribund fish ranged between 1.7 × 10³ to 1.6 × 10⁵ CFU/50 μL whole blood. Clearance of bacteria from the blood was observed by 216 h post-exposure and all fish surviving bacterial exposure developed agglutinating antibody against E. ictaluri . The pathogenesis of the infection was accompanied by the shedding of viable E. ictaluri into the water which may serve as a mechanism by which fish to fish transmission occurs.     

黄颡鱼甘露糖受体基因的克隆和功能分析

甘露糖受体(MR)隶属凝集素超家族,主要表达于巨噬细胞和未成熟的树突状细胞表面。MR不仅在先天免疫防御中发挥重要作用,还通过参与抗原呈递,激活T淋巴细胞,启动获得性免疫应答过程。本研究采用同源克隆技术获得黄颡鱼甘露糖受体(pf MR)基因,采用荧光定量PCR技术检测MR在正常黄颡鱼体内的分布情况,采用鲖爱德华菌感染黄颡鱼头肾巨噬细胞和甘露聚糖封闭MR方法研究黄颡鱼MR在抗细菌感染中的作用。结果显示,pf MR同团头鲂、草鱼、斑马鱼和尼罗罗非鱼的MR聚为一支。pf MR在所检测的12个组织中均有分布,其在肾脏、脾脏和肌肉组织中表达量较高,在血液中表达量较少。鲖爱德华菌感染黄颡鱼头肾巨噬细胞后,pf MR、IL-1β和TNF-a均被细菌诱导表达,超氧阴离子和一氧化氮的含量也上升,超氧阴离子在感染30 min后即显著上升,一氧化氮在感染12 h后才显著上升。甘露聚糖竞争结合MR,显著抑制巨噬细胞内化GFP标签的鲖爱德华菌,加入EDTA减少内化的荧光强度,加入Ca~(2+)使内化的荧光强度回升。研究表明,黄颡鱼头肾巨噬细胞MR参与鲖爱德华菌的识别和内吞过程,而且依赖Ca~(2+)。     

Recovery and Viability of Edwardsiella ictaluri from Great Blue Herons Ardea herodias Fed E. ictaluri-Infected Channel Catfish Ictalurus punctatus Fingerlings

Feeding activities of great blue herons Ardea herodias in catfish ponds during outbreaks of enteric septicemia of catfish have been implicated as a mechanism for the transmission of the disease from infected to uninfected ponds. Although Edwardsiella ictaluri , the causative agent, has been identified in gastrointestinal tracts of great blue herons, the role of these birds as a vector of E. ictaluri is not well documented. The potential of these birds to contaminate catfish ponds with E. ictaluri was investigated by feeding captive herons over a 4-d period with catfish fingerlings injected intraperitoneally with live E. ictaluri . Daily fecal samples, throat and rectal swabs, and feather samples were collected, cultured and examined for E. ictaluri using both a selective media and a monoclonal indirect fluorescent antibody test specific for E. ictaluri . Gastrointestinal tracts sampled at the conclusion of the feeding trial were similarly examined. While E. ictaluri was detected using the indirect fluorescent antibody test, no viable E. ictaluri was cultured from either feces, gastrointestinal tracts or feathers. Growth of E. ictaluri was not observed at 40 C, the rectal temperature observed in captive great blue herons. Prior incubation at 40 C suppressed the growth of E. ictaluri at 24 C, an optimal temperature for growth of this bacterium. These results indicate that great blue herons appear to play little or no role in the transmission of E. ictaluri among catfish ponds.     

Organization and sequence of four flagellin-encoding genes of Edwardsiella ictaluri

Edwardsiella ictaluri , the cause of enteric septicaemia in channel catfish ( Ictalurus punctatus ), is motile by means of peritrichous flagella. We determined the complete flagellin gene sequences and their organization in E. ictaluri by sequencing genomic segments from a λ-ZAP phage genomic library of E. ictaluri . Four flagellin genes ( fliC1, fliC2, fliC3 and fliC4 ) are arranged in tandem within 6 kb in the E. ictaluri genome. Each flagellin-coding sequence is preceded by a σ²⁸ recognition site consensus sequence. The predicted amino acid sequences of all four flagellin proteins (between 36 and 37.5 kDa) are similar in the N-terminal (1–160 aa) and C-terminal (last 74 aa) portions and are divergent in the central portion of the proteins. Proteins encoded by flC1, fliC2 and fliC3 are more similar to each other (88–90% aa identity) than to the protein encoded by fliC4 (76–78% aa identity). basic local alignment search tool analysis of GenBank sequences showed that all flagellin aa sequences are more similar to those of Serratia marcescens (72–74% identity) than to those of Edwardsiella tarda (≤64% identity). Primary determination of E. ictaluri flagellin gene sequences facilitate advanced studies on the role of flagella in host–pathogen interaction.     

Growth Response and Resistance to Edwardsiella ictaluri of Channel Catfish,Ictalurus punctatus,Fed Diets Containing Distiller’s Dried Grains with Solubles

A study was conducted to examine the effect of dietary levels of distiller’s dried grains with solubles (DDGS) on growth, body composition, hematology, immune response, and resistance of channel catfish, Ictalurus punctatus, to Edwardsiella ictaluri challenge. Five diets containing 0, 10, 20, 30, and 40% DDGS with supplemental lysine (Diets 1–5) as partial replacements of a combination of soybean meal and cornmeal on an equal protein basis were fed to juvenile catfish (13.33 ± 0.25 g) for 12 wk. Growth performance and feed utilization efficiency were similar for fish in all treatments. Body lipid and moisture increased and decreased, respectively, in fish feed DDGS‐containing diets relative to the control group. Dietary treatment had no effect on red and white blood cell counts. Hemoglobin and hematocrit were significantly higher in fish fed diets containing DDGS than in those fed the control diet. Fish fed 20–40% DDGS diets had increased serum total immunoglobulin, and those fed the 30% DDGS diet had significantly increased antibody titers 21 d following E. ictaluri challenge. Other immune variables evaluated were not affected by dietary treatments. Preliminary results on bacterial challenge showed an increased resistance against E. ictaluri in fish fed DDGS‐containing diets (Diets 2–5).     

Comparative Susceptibility of Channel Catfish,Ictalurus punctatus; Blue Catfish,Ictalurus furcatus; and Channel (♀) × Blue (♂) Hybrid Catfish to Edwardsiella piscicida,Edwardsiella tarda,and Edwardsiella anguillarum

Members of the genus Edwardsiella are important pathogens of cultured and wild fish globally. Recent investigations into the phenotypic and genotypic variation of Edwardsiella tarda have led to the segregation of E. tarda into three distinct taxa: E. tarda, Edwardsiella piscicida, and Edwardsiella anguillarum. In catfish aquaculture in the southeastern USA, E. piscicida has been more commonly associated with disease than E. tarda or E. anguillarum, and recent research has demonstrated E. piscicida to be more pathogenic in channel catfish than E. tarda or E. anguillarum. Anecdotal reports from industry suggest an increased prevalence of E. piscicida associated with the culture of channel (♀) × blue (♂) hybrid catfish. This work investigated the comparative susceptibility of channel catfish, blue catfish, and their hybrid cross to molecularly confirmed isolates of E. tarda, E. piscicida, and E. anguillarum. There was significantly higher mortality in hybrid catfish compared to channel catfish following intracoelomic injection of E. piscicida. To our knowledge, E. piscicida is the first bacterial pathogen to demonstrate increased pathogenicity in hybrid catfish compared to channel catfish.     

Protective immunity against enteric septicaemia in channel catfish, Ictalurus punctatus (Rafinesque), following controlled exposure to Edwardsiella ictaluri

Protective immunity against enteric septicaemia of catfish (ESC) following immunization with Edwardsiella ictaluri bacterins and exposure to live E . ictaluri was investigated. Mean cumulative percentage survival was significantly higher ( P 0.05) in controlled live vaccinates (100%) than in immersion and oral bacterin vaccinates (68.3% and 50.0%, respectively). Bactericidal activity against E . ictaluri by peritoneal macrophages from controlled live vaccinates (85.9%) was significantly greater ( P 0.05) than bactericidal activity of macrophages from immersion bacterin vaccinates (71.4%) or non-vaccinates (68.1%). No significant ( P > 0.05) difference was found in the bactericidal activity of macrophages from oral bacterin vaccinates and macrophages from controlled live vaccinates. The E . ictaluri -specific antibody response of controlled live (0.08 OD) and immersion bacterin vaccinates (0.11 OD) was significantly higher ( P 0.05) than that of oral bacterin vaccinates and non-vaccinates (0.01 OD) 15 days post-vaccination. A significantly higher antibody response was seen in controlled live vaccinates (0.17 OD), when compared to other vaccinates or non-vaccinates 33 days after vaccination. Neither immersion nor oral bacterins protected the vaccinates against ESC. Controlled live E . ictaluri immunization of channel catfish resulted in production of specific antibodies, increased macrophage bactericidal activity and protection against ESC.     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

Cross-protection elicited in channel catfish (Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri strains

Cross-protection of channel catfish ( Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri and challenged with six E. ictaluri strains was examined in four trials. The relative per cent survival among low-dose immunized and then challenged fish ranged from 27.7% to 100%. Significant protection ( P <0.05), with the exception of strain ATCC-33202, was conferred by immunization with a given E. ictaluri strain challenged either with a homologous or a heterologous strain. Antibody titres of pooled serum collected on day 22 from surviving fish examined by enzyme-linked immunosorbent assay (ELISA) ranged from 1:40 to 1:320, but no differences were apparent among different vaccinated groups. The protein profiles of six E. ictaluri strains examined by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a relatively homogeneous pattern. Immuno-blots probed with pooled serum from immunized and challenged fish showed a pattern similar to LPS-reaction patterns observed with E. ictaluri in other studies. Since the present studies further corroborate that E. ictaluri is a clonal bacterial species with no apparent antigenic differences, it is possible that immunization with a single E. ictaluri field strain should confer protection against any other strain.     

Effect of Feeding Frequency and Romet-Medicated Feed on Survival, Antibody Response, and Weight Gain of Fingerling Channel Catfish Ictalurus punctatus after Natural Exposure to Edwardsiella ictaluri

Abstract The efficacy of restrictive feeding regimes and Romet®-medicated feed (formulated at 40.5-mg Romet-30 premix/kg feed) fed at 2.0% body weight was evaluated under field trial conditions for the treatment of naturally induced Edwardsiella ictaluri infections in fingerling channel catfish. After detection of an Edwardsiella ictaluri epizootic, fish were fed medicated and non-medicated feed every day, every other day and every third day for the duration of the experiment. In addition, experimental treatments also included fish that were completely withheld from feed and those provided Romet-medicated feed for five consecutive days followed by non-medicated feed fed daily. Feeding frequency and diet significantly affected survival and weight gain. Survival was greatest among fish that were completely withheld from feed or that were fed medicated feed every other or every third day. The use of non-medicated feed every other or every third day was equally as effective for reducing Edwardsiella ictaluri associated mortalities as feeding Romet-medicated feed on a daily basis. Feeding fish Romet-medicated feed for five consecutive days was not an effective treatment. Agglutinating antibody titers against Edwardsiella ictaluri were significantly lower in all groups of fish fed Romet-medicated feed for the duration of the experiment. These data indicate that feeding frequency is a primary factor affecting the severity of Edwardsiella ictaluri infection and that prolonged consumption of Romet-medicated feed may suppress antibody production.     

抗黄颡鱼“红头病”功能性蛋粉的制备及其应用

为了评价抗爱德华氏菌(Edwardsiella ictaluri)的特异性Ig Y的功能性蛋粉在黄颡鱼(Pelteobagrus fulvidraco)"红头病"预防及治疗中的应用效果,采用灭活的爱德华氏菌(菌种保藏号:CCTCCNO:M2012024)免疫蛋鸡,通过微包膜及高速离心喷雾干燥技术制备抗爱德华氏菌的特异性Ig Y的功能性蛋粉,采用泼洒和口服两种方式检测该功能性蛋粉对黄颡鱼的免疫保护率。结果显示,所制备的功能性蛋粉抗体效价达到1∶5120;以剂量为2 mg/L功能性蛋粉连续泼洒水体3 d或以饲料重量0.2%的剂量口服14 d后,再用浓度为2.0×107CFU/m L的致病性爱德华氏菌对健康黄颡鱼进行攻毒,泼洒和口服功能性蛋粉对黄颡鱼的免疫保护率分别为85.5%和70.0%;对人工感染爱德华氏菌后患病的黄颡鱼养殖水体连续泼洒5 d或投喂14 d功能性蛋粉,其对黄颡鱼的免疫保护率分别为67.2%和40.7%,表明制备的功能性蛋粉对黄颡鱼"红头病"有很好的预防和治疗效果。     

Influence of the Dietary Level of Iron from Iron Methionine and Iron Sulfate on Immune Response and Resistance of Channel Catfish to Edwardsiella ictaluri

Channel catfish Ictalurus punctatus fingerlings were fed purified diets supplemented with iron at levels of 0, 20, 60, and 180 mg/kg from iron sulfate (FeS) or 5, 10, 20, 60, and 180 mg/kg from iron methionine (FeM) in triplicate tanks for 8 wk. Fish were then divided into two groups and subjected to different assays to measure disease resistance and individual immune functions. Representative fish from each dietary treatment were challenged by bacterial immersion with virulent Edwardsiella ictaluri , and mortality due to enteric septicemia was recorded. Other fish were immunized with 0.2–mL formalin-killed E. ictaluri and boosted 21 d post-immunization. Antibody response was determined by FAST-ELISA. Chemiluminescent and chemotaxis assays were performed using peritoneal macrophages. Supplementation of the diet with various levels of iron from FeS or FeM did not significantly affect antibody production. Chemotactic migration by macrophages was depressed in iron-deficient fish and a level of 60 mgkg from either FeS or FeM provided the highest chemotactic indexes. A deficiency of dietary iron was found to increase mortality of channel caffish due to enteric septicemia of catfish (ESC). However, more studies should be conducted to better understand the effects of sources and levels of dietary iron on immune responses and disease resistance in channel caffish.