Molecular cloning and sequencing of hemoglobin-β gene of channel catfish, Ictalurus punctatus Rafinesque

The hemoglobin-β gene of channel catfish, Ictalurus punctatus, was cloned and sequenced. Total RNA from head kidneys was isolated, reverse transcribed and amplified. The sequence of the channel catfish hemoglobin-β gene consists of 600 nucleotides. Analysis of the nucleotide sequence reveals one open reading frame and 5′- as well as 3′-untranslated regions. The open reading frame of the sequence potentially encodes 148 amino acids with a calculated molecular mass of 16.3 kDa. The pI and charge at pH 7.0 of the deduced hemoglobin-β protein were 7.28 and 0.47, respectively. Overall, 22 amino acid residues were conserved throughout the sequences, including His64 and His93, the sites for heme-binding. Unlike the counterpart of other common cultured fish such as Salmo salar, Oncorhynchus nerka, Oncorhynchus mykiss, Cyprinus carpio and Ctenopharyngodon idella, the hemoglobin-β of channel catfish did not have cysteine. The amino acid sequence of channel catfish hemoglobin-β shows 84% homology with that of Silurus asotus (both are in the order Siluriformes). However, comparison with those of other fish species shows homology ranging from 53 to 68%. Structural analysis by the 3D-PSSM program displays that channel catfish hemoglobin-β has eight α-helices, A–H.     

Susceptibility of channel catfish, Ictalurus punctatus (Rafinesque), to Edwardsiella ictaluri challenge following copper sulphate exposure

Channel catfish, Ictalurus punctatus (Rafinesque), with or without a preliminary 24 h exposure to 2 mg copper sulphate L(-1), were challenged with 7.5 x 10(6) colony forming units L(-1) of Edwardsiella ictaluri to determine the effect of copper sulphate on disease resistance. Catfish previously exposed to copper sulphate were significantly more resistant to the bacterial challenge than those not exposed. Catfish not exposed to copper sulphate suffered 35.5% mortality while catfish exposed to copper sulphate experienced 14.1% mortality. Copper concentrations were the same in tank waters of both exposed and control fish at the time of challenge, eliminating the possibility that copper in the water may have been toxic to bacteria. Copper concentrations in freeze dried and ground tissues of unexposed, exposed, and purged channel catfish were highest in fish before copper sulphate exposures suggesting that elevated tissue levels of copper were not responsible for the increased resistance to bacterial challenge. Competition for sites of bacterial attachment to gill or epithelial cells may account for the reduction in mortality; although this is not supported by the low copper content of fish tissue after copper exposure.     

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.     

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.     

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.     

The effect of feeding diets containing deoxynivalenol contaminated corn on channel catfish (Ictalurus punctatus) challenged with Edwardsiella ictaluri

Channel catfish were fed practical corn‐soybean meal diets for 10 weeks that contained various weighed amounts of ground, dried field corn contaminated with 20 mg deoxynivalenol (DON) kg⁻¹. Weighed amounts of DON corn were blended with weighed amounts of ground, clean corn that contained no DON (0 mg kg⁻¹) to yield five diets that had 0, 2.5, 5.0, 7.5 and 10.0 mg DON kg⁻¹ of diet. Results show that catfish fed diets that contained DON for 7 weeks did not experience lower weight gains or poorer feed conversion ratios that were significantly (P > 0.05) different from control‐fed fish. Mortality of catfish during the 21‐day post‐challenge period indicate that catfish fed diets containing DON‐contaminated corn that provided at least 5.0 mg DON kg⁻¹ of diet had significantly (P < 0.05) lower mortality than catfish fed the control diet or the diet that provided 2.5 mg DON kg⁻¹ of diet. The presence of DON‐contaminated corn in the experimental diets did not significantly (P > 0.05) alter fish body weight gains and appeared to provide a protective effect for channel catfish challenged with the pathogenic bacterium Edwardsiella ictaluri.     

Re-evaluation of vitamin E supplementation of practical diets for channel catfish, Ictalurus punctatus, production

A 10-month feeding trial was performed with channel catfish, Ictalurus punctatus , to re-evaluate the minimum level of dietary vitamin E supplementation required under conditions of commercial production. Four levels (0, 15, 30 and 60 mg kg⁻¹ diet) of supplemental vitamin E were added as DL -α-tocopherol acetate to a typical commercial catfish diet. Sixteen 0.04-ha ponds were stocked in July, at a rate of 25 000 fish ha⁻¹, with two size classes (averaging 18 and 265 g fish⁻¹) of fingerling channel catfish, and each diet was fed to fish in four replicate ponds once daily to satiation. After 10 months of feeding, a total harvest was performed to obtain final production data, and samples were obtained from fish in all ponds. Liver and plasma samples were obtained for α-tocopherol determination, and liver, heart and muscle samples were processed for histological evaluation.
No discernible differences occurred among fish fed the different diets with respect to weight gain or survival. Mean production rate was 9734 kg ha⁻¹, with a final average fish weight of 0.53 kg. A significant ( P < 0.05) effect of diet was noted in plasma and liver α-tocopherol levels, which increased with dietary supplementation. An effect of size also was apparent, with plasma and liver α-tocopherol levels being higher in larger fish compared with smaller fish within a dietary treatment. Histological evaluation revealed no differences among fish fed the different dietary treatments. Based on the lack of overt histological signs of deficiency, it appears that the current level of vitamin E supplementation of commercial catfish diets may be reduced considerably with no detriment to channel catfish health or production.     

Evaluation of potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

An experiment was performed to evaluate the efficacy of potassium permanganate (KMnO₄) as a prophylactic and therapeutic treatment of an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Fish were cutaneously abraded and divided into five treatment groups: (i) challenged by waterborne exposure to F. columnare and not treated with KMnO₄ (positive control), (ii) challenged and simultaneously treated with KMnO_4, (iii) challenged and treated with KMnO₄ at 1, 6 and 9 days post-challenge, (iv) not challenged and treated with KMnO₄ at 1, 6 and 9 days post-challenge (first negative control) and (v) not challenged and not treated (second negative control). The dosing of KMnO₄ was 2.0 mg L⁻¹ above the potassium permanganate demand for 2 h duration. The survival of the group challenged and simultaneously treated with KMnO₄ (99%) was significantly higher than the positive control (78%) and was not significantly different from the negative control groups. The challenged fish treated with KMnO₄ post-challenge had 7% higher survival than the positive control (85% compared with 78%), but that difference was not statistically significant. The results demonstrate that KMnO₄ has a clear prophylactic value but probably a marginal therapeutic value once the infection has established.     

不同商品饲料对生长、体色及肝脏组织结构的影响

本试验评估了(Ictalurus punctatus)主产区4种商品颗粒饲料(分别记为S1、S2、S3和S4,其中S4为膨化饲料,其他均为颗粒饲料)和自行设计的颗粒饲料(记为S5)对试验阶段生长性能、体色变异、肝胰脏组织结构及形态指标的影响。7周的饲喂试验结果显示:饲料蛋白含量为38%的S4膨化饲料可明显的提高养殖鱼的体重,平均增重率为262.6%,但存活率较低,仅为72.3%;而喂养S5饲料组的增重率为168.89%,且存活率为92.4%,高于其他饲料组。养殖结束后,不同试验组的肥满度、脏体比有所差异,但均未达到显著性水平(P>0.05)。投喂S2饲料组养殖鱼体体色发白比较明显,体色异常比例高达91%;S4饲料组养殖后体色发白也比较明显,体色异常比例为72.3%;对背部黑色素扫描研究进一步证实饲喂S2饲料和S4饲料的黑色素细胞发生了变化,黑色素细胞边缘变得模糊,而自配S5饲料的黑色素细胞树突状分支较多;饲喂S4饲料组的背部斑点比例为12.15%,而S2饲料组的背部斑点比例为26.38%,自配S5饲料组的背部斑点比例为35.69%,S4饲料组的斑点比例明显较低。     

富硒酵母对斑点叉尾鮰肝脏的毒性作用及蛋白质组学的影响

实验探讨了富硒酵母对斑点叉尾鮰肝脏的毒性作用及蛋白质组学的影响,结果表明,饲养56 d后正常组和有机硒组采用原子荧光法检测硒含量均值为0.15和0.81 mg/kg。H.E染色显示有机硒组肝细胞有明显的疏松化,部分发生脂肪变。通过二维凝胶电泳结合ImageMaster软件分析,鉴定出8个表达差异蛋白质点,其中3个在有机硒组表达上调,5个在有机硒组特异表达,经液相色谱串联质谱分析鉴定,这些蛋白质分别是伴侣蛋白TCP1-亚基8、甘油醛-3-磷酸脱氢酶、4SNcTudor蛋白、腺苷激酶、酮糖移转酶、丙氨酰-tRNA合成酶。富硒酵母饲料对斑点叉尾鮰肝具有明显的毒性效应,有机硒调控4SNc-Tudor蛋白,通过信号传导通道加强机体免疫。     

鳗源迟缓爱德华氏菌菌蜕的构建及制备条件优化

菌蜕具有完整细菌表面抗原结构, 可诱导机体的体液和细胞免疫应答, 成为疫苗制备的候选之一,为了探讨鳗鲡迟缓爱德华氏菌菌蜕疫苗的可行性, 实验采用基因重组技术构建了噬菌体PhiX174裂解酶基因(Lysis E)的温控表达载体, 转化迟缓爱德华氏菌, 成功制备其菌蜕, 并对菌蜕形态、溶菌动力学、裂解效率以及制备条件等进行了研究。结果表明, 细菌菌蜕表面形成溶菌孔道, 细胞因内容物流失而发生明显的皱缩; 构建的迟缓爱德华氏菌诱导后1 h开始裂解, 5 h后裂解基本完成, 裂解效率为99.99%, 冷冻干燥后重悬涂布平板, 未检出活菌, 电镜观察表明冻干前后细胞形态未见明显变化; 构建的迟缓爱德华氏菌分别在OD₆₀₀值为0.4和0.6进行诱导, 其裂解过程和裂解效率没有明显区别; 分别用LB、BHI、NB 3种培养基进行比较研究, 其中LB培养基制备的菌蜕细胞较完整、裂解完全, 是制备迟缓爱德华氏菌菌蜕最优培养基。本研究成功构建了鳗源迟缓爱德华氏菌菌蜕, 并对其制备条件进行了优化, 为鳗鲡爱德华氏菌病疫苗开发奠定了基础。     

Edwardsiella tarda infection in Korean catfish,Silurus asotus,in a Korean fish farm

Mass mortality of Korean catfish, Silurus asotus, occurred in a culture farm situated in Jeollabukdo Province, Korea. The cumulative mortality rates reached up to 5% of the total fish in the farm per day. In clinical signs, the affected fish showed abdominal distension, vent protrusion, enteritis, liver congestion and abscess‐like lesions in enlarged spleen and kidney. Histopathologically, in the liver, hepatocytes lost fat and underwent atrophy or necrosis. The spleen showed necrotized splenocytes and a haemorrhagic pulp. In the kidney, glomerular destruction, degeneration of renal tubular epithelial cell and haemorrhage were observed. However, necrotic muscular lesions were not observed. A pure bacterial isolate was obtained from the liver, spleen and kidney lesions of affected fish. Experimental infection of normal catfish with the isolate resulted in the development of clinical signs similar to those seen on the farm. The isolates were identified as Edwardsiella tarda through biochemical tests (99.4%) and analysis of bacterial genes (16S rDNA) sequences (98%). The bacteria possessed two virulent genes: sodB and katB genes. These results suggest that E. tarda can act as a pathogen of farmed catfish. This is the first report showing that E. tarda caused mortality in cultured Korean catfish.     

饲料低蛋白、高脂肪和高消化能对斑点叉尾鮰

为了探讨高脂肪条件下不同的蛋白和能量水平对斑点叉尾鮰生长及体组成的影响,试验设2个蛋白水平(22%,28%),2个脂肪水平(10.0%,14.0%),2个消化能水平(12.56 kJ/g,14.23 kJ/g),共8组,分别为P28L10E14.23,P28L14E14.23,P22L10E14.23,P22L14E14.23,P28L10E12.56,P28L14E12.56,P22L10E12.56和P22L14E12.56。试验饲料配方使用鱼粉和豆粕调节蛋白含量,混合油脂(鱼油∶玉米油=1∶1)调节脂肪含量,α-淀粉、次粉和麸皮调节消化能含量,并以微晶纤维素为填充物,每组3个重复,每个重复20尾鱼(141.5?1.0) g。饲养60 d后,进行生产性能测定,并采集组织样本,测定相关指标。结果表明,鱼体末重(FW)、增重率(WG)、特定生长率(SGR)和饵料系数(FCR)不受饲料蛋白、脂肪和消化能单一营养水平影响(P>0.05);但与饲料蛋白、脂肪和消化能三者的交互作用(P<0.01)有关。胴体蛋白、脂肪含量与饲料脂肪和消化能水平相关(P<0.05)。由此可见,饲料过高脂肪和消化能对斑点叉尾鮰没有额外的促生长作用;饲料蛋白水平达到22%时,即可满足140~300 g斑点叉尾鮰的营养需要;饲料中22%的蛋白、10%的脂肪、12.56 kJ/g的消化能即可满足斑点叉尾鮰仔鱼的生长需要,同时又能保证其正常生理机能,建议可在实际生产配方中作参考值。