黄颡鱼鲇爱德华氏菌的分离鉴定及其致病性研究

从患头顶溃疡症的黄颡鱼(Pelteobagrus fulvidraco)内脏组织中分离出致病力强的菌株JZ086,对该菌进行了形态学、生理生化特性的测定,并进行了Biolog全自动微生物分析系统及16S rDNA序列鉴定。结果显示:人工感染实验证实该菌为黄颡鱼的病原菌,其半数致死量(LD50)为1.7×105CFU。Biolog全自动微生物分析系统鉴定结果显示该菌为鲇爱德华氏菌(Edwardsiella ictaluri)。16S rDNA序列分析显示该菌与鲇爱德华氏菌的亲缘关系最近,同源性高达99%;系统进化树中与鲇爱德华氏菌(AB050826)自然聚为一支,二者遗传距离约为0.002。鉴定结果确认JZ086为鲇爱德华氏菌。药物敏感性试验结果显示,该菌对所测试的15种药物都敏感,其中对氨苄西林、环丙沙星、左氧氟沙星、妥布霉素等尤其敏感。     

感染累枝虫对黄颡鱼血液生化指标的影响

为探究感染累枝虫(Epistylis sp.)的黄颡鱼(Pelteobagrus fulvidraco)病愈过程中血液生化指标的变化规律,测定了患病鱼和经药物治疗痊愈及健康的黄颡鱼的血清皮质醇、葡萄糖、乳酸(LD)含量以及碱性磷酸酶(AKP)、乳酸脱氢酶(LDH)、谷草转氨酶(AST)、谷丙转氨酶(ALT)的活性。结果显示,患病组黄颡鱼血清皮质醇、血糖含量和AST活性均显著高于痊愈组(P0.05),血清ALT活性与痊愈组无显著差异(P0.05)。与患病组相比,痊愈组黄颡鱼血清LD含量,LDH和ALP活性均显著升高(P0.05)。与健康组相比,痊愈黄颡鱼血清皮质醇含量和ALT活性无显著差异(P0.05),而葡萄糖、LD含量,LDH、AKP和AST活性则差异显著(P0.05)。结果表明,累枝虫的寄生引起了黄颡鱼显著的应激反应,使鱼体非特异性免疫能力下降;经治疗痊愈后的黄颡鱼的非特异性免疫力可恢复正常,鱼体应激反应消失并表现出正常的游泳行为。     

一株黄颡鱼源迟缓爱德华氏菌弱毒株的分离鉴定及特性分析

为探明湖北荆州市某养殖基地患病黄颡鱼(Pelteobagrus fulvidraco)以烂身、腹水为主要症状且持续性死亡的确切病因,本研究对患病黄颡鱼进行了病原筛查,从患病黄颡鱼组织中分离到一株优势菌,经过形态特征、生理生化鉴定、16S rRNA测序和系统进化分析确定分离菌株为迟缓爱德华氏菌,命名为Et-4.结果显示:分离菌株Et-4 具有较强的生物被膜形成能力,不携带esaV、fimA、gadB和katB四种主要的毒力基因.分离菌株Et-4人工感染健康黄颡鱼出现与自然发病类似的症状,并能从病鱼中再次分离出该菌,证实迟缓爱德华氏菌是引起此次黄颡鱼持续性死亡的致病原;分离菌株Et-4 对黄颡鱼的LD50为 3.9×106 CFU/g,结合毒力基因谱判定其为弱毒株.分离株Et-4 携带耐药基因tet A、sul Ⅰ和add A1,对头孢类药物、庆大霉素、新霉素等敏感;对四环素类、青霉素类、万古菌素等耐药;中药抑菌实验表明乌梅和丁香对迟缓爱德华氏菌Et-4 有明显的抑制效果.     

黄颡鱼红头病病原研究

从患红头病的黄颡鱼的肝脏中分离获得HS807菌株,经人工感染试验为该病的致病菌,结合形态观察、培养特性及16S rRNA基因序列分析,HS807菌为鲇鱼爱德华氏菌.药敏试验表明,对复方新诺明、环丙沙星、先锋Ⅳ、强力霉素、青霉素、依诺沙星、菌必治、新霉素、呋喃唑酮等药物敏感.     

黄颡鱼精子入卵的扫描电镜观察

应用扫描电镜观察了黄颡鱼(Pelteobagrus fulvidraco)成熟卵和精子的形态、卵子对精子的应答反应、受精孔的数量与形态、受精方式与过程。结果显示:黄颡鱼精子为鞭毛型,头部近圆球形,无顶体,单尾;卵为圆球形,卵膜表面多嵴,且有15～16条宽度不同的小沟;卵表面仅有1个受精孔,精孔管口内径约2.8μm。黄颡鱼为单精受精,受精后30 s内完成精子入卵过程。     

黄颡鱼吻端细胞培养研究

采用M199培养基对黄颡鱼(Pelteobagrus fulvidraco)吻端组织进行体外培养研究,继代培养细胞传至第8代。实验结果显示:吻端组织接种培养3~5 h,细胞迁移伸展,6 d后生长细胞集落汇合,每4~6 d递次进行继代培养,培养生长的细胞呈纤维样。比较继代细胞在不同温度(15、27、37℃)和血清浓度(0、10%、20%)条件下的生长,27℃、10%血清的培养条件最适宜吻端细胞的增殖。通过染色体组型分析,证明离体吻端细胞为正常的二倍体细胞。研究结果表明,黄颡鱼离体吻端细胞对温度等培养条件有很强的适应性,培养细胞增殖迅速,具有建立连续细胞系的潜力。     

低渗溶液浓度对黄颡鱼精子活力和受精率的影响

为提高黄颡鱼(Pelteobagrus fulvidraco)人工授精的稳定性,分别用卵子和精子同步激活法以及预激活精子法,在6个梯度浓度(0%～0.5%)范围的低渗溶液中进行人工授精试验,比较精子活力、受精率和孵化率。结果显示:低渗溶液浓度与精子活力、精子激活率和受精率密切相关,在相同浓度的低渗溶液组中采用预激活精子法的受精率显著高于卵子和精子同步激活法(P<0.05),受精率相对应提高了7.7%～14.5%;低渗溶液的浓度和人工授精方式对孵化率无显著影响。结果表明在浓度0.3%的低渗溶液中采用预激活精子法的效果最好。     

柱形病对黄颡鱼血液生化指标的影响

为探究柱形病对黄颡鱼(Pelteobagrus fulvidraco)生理机能的影响,随机选取患病和健康黄颡鱼各10尾,采集其血液,静置后离心分离血清以测定血液生化指标如皮质醇、乳酸(LD)含量,乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)活性,葡萄糖、白蛋白(ALB)、总蛋白(TP)、尿素氮(BUN)、总胆固醇(TCHO)、高密度脂蛋白胆固醇(HDLC)、低密度脂蛋白胆固醇(LDLC)含量,谷草转氨酶(AST)、谷丙转氨酶(ALT)活性。结果显示:患病黄颡鱼血清皮质醇浓度和LDH、AST活性均极显著高于健康鱼,LD含量和ALT活性显著高于健康鱼。与健康鱼相比,患病黄颡鱼血清葡萄糖含量极显著降低,而HDLC、LDLC、TCHO、ALB、TP、BUN浓度和ALP活性无显著差异。健康鱼肝细胞呈近圆形,形状较规则,细胞界线清晰,细胞核多位于细胞中央,呈圆形或椭圆形,肝细胞以中央静脉为中心形成放射状排列。患病鱼肝细胞出现局部坏死、空泡化,形成坏死灶,伴有大量中性粒细胞、淋巴细胞浸润。结果表明:柱状黄杆菌感染可引起黄颡鱼鱼体显著的应激反应,并引起鱼体肝脏损伤。     

黄颡鱼暴发性流行病病原的分离鉴定及其3种毒力基因检测

为查明黄颡鱼(Pelteobagrus fulvidraco)暴发性流行病的病原菌及其3种毒力基因的携带情况,以常规方法进行细菌分离,人工感染试验确定病原菌的致病性,API 20NE和16S rRNA基因序列分析进行细菌鉴定,PCR扩增法检测病原菌的3种毒力基因。结果显示,从发病症状典型的黄颡鱼肝脏中分离到1株嗜水气单胞菌(Aeromonas hydrophila)GXGP1,与嗜水气单胞菌标准株ATCC 7966T的同源性为99.9%,对黄颡鱼有很强致病力,是引起黄颡鱼暴发性流行病的病原菌,该菌携带有Aer、hly和ahp 3种毒力基因。     

Isolation and characterization of Edwardsiella ictaluri strains as pathogens from diseased yellow catfish Pelteobagrus fulvidraco (Richardson) cultured in China

Yellow catfish Pelteobagrus fulvidraco (Richardson) is a commercially important fish generally distributed in Southeast Asian countries. The well‐known aetiological agent of enteric septicaemia of catfish, Edwardsiella ictaluri, was isolated from diseased yellow catfish P. fulvidraco (Richardson) reared at two commercial fisheries in China. The economic losses due to the high mortalities (about 50%) caused by this bacterium have been increasing annually. The affected fish presented two different, typical symptoms: pale gills, slight exophthalmia and a ‘hole in the head’, and haemorrhage on the opercula, in the skin under the jaw, creating a ‘hole under the jaw’. These diseases were found frequently in cultured yellow catfish throughout China. The isolates from both outbreaks were all Gram negative, facultatively anaerobic and short rod. Morphological and biochemical tests and phylogenetic analysis based on the 16S rDNA sequences all strongly indicated that these yellow catfish isolates were highly identical to the known E. ictaluri. In addition, the isolates possessed the typical plasmid profile of E. ictaluri. Experimental infection assays were conducted and pathogenicity (by an intraperitoneal injection) was demonstrated in yellow catfish and channel catfish Ictalurus punctatus. The results showed that yellow catfish isolates were quite conservative phenotypically and genetically, and were able to cause two different, typical symptoms in this fish under unknown conditions and mechanism.     

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.     

皮质醇混饲投喂对黄颡鱼幼鱼生长及外周血细胞的影响

采用皮质醇混饲投喂的试验方法,模拟自然条件下的慢性应激,观察和分析了应激激素皮质醇对黄颡鱼(Pelteobagrus fulvidraco)幼鱼生长和外周血细胞的效应。试验组饲料中皮质醇添加剂量为100 mg/kg,日投饵率为3%,试验期5周。结果表明:(1)外源皮质醇通过减少肠道黏膜皱褶和促进糖异生作用使黄颡鱼幼鱼增重受阻;(2)外源皮质醇使黄颡鱼幼鱼外周血白细胞数量显著减少、淋巴细胞百分比显著降低,但是中性粒细胞和单核细胞百分比则呈上升趋势;(3)外源皮质醇能促进红细胞增生,增加黄颡鱼幼鱼外周血红细胞数量。     

复方中草药制剂对黄颡鱼长和非特异性免疫功能的影响

在基础饲料中添加0.5%、1.0%、2.0%的复方中草药制剂,连续投喂60d后,通过测定供试黄颡鱼的生长性能、血细胞的吞噬活性、血清溶菌酶活性,比较了复合中草药制剂对黄颡鱼促生长及非特异性免疫功能的影响。结果显示:饲料中添加0.5%的复方中草药制剂对黄颡鱼的生长影响显著(P0.05),添加1.0%和2.0%的中草药制剂对黄颡鱼的血细胞的吞噬活性、溶菌酶活力影响极显著(P0.01)。试验组可显著提高黄颡鱼生长、血细胞的吞噬活性、溶菌酶活力。     

A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

The most common bacterial diseases in pond‐raised channel catfish Ictalurus punctatus (Rafinesque) are enteric septicemia of catfish and columnaris, caused by Edwardsiella ictaluri and Flavobacterium columnare respectively. Medicated feed containing antibiotics is one management approach that catfish producers use in the treatment of bacterial diseases. However, the future use of all types of medicated feed in catfish aquaculture is uncertain. To discover effective alternatives to antibiotics, a rapid 96‐well microplate bioassay utilizing E. ictaluri and F. columnare to evaluate natural compounds and extracts was developed. In this bioassay, bacterial growth is determined by absorbance measurements of microplate wells after 24 h incubation and then confirmed by detecting cell viability after the addition of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide with additional incubation for 24 h. The minimum inhibitory concentration, minimum bactericidal concentration and 50% inhibition concentration (IC50) are determined by graphing the absorbance data. The 24 h IC50 results of test compounds are compared with the 24 h IC50 results of the drug controls oxytetracycline and florfenicol. Among the antibiotics evaluated, doxycycline and tetracycline appear more effective against E. ictaluri and F. columnare than either drug control. This bioassay is rapid, reproducible and economical for evaluating a large number of compounds and extracts.     

黄颡鱼规模化养殖高产试验

黄颡鱼对生态环境的适应能力较强,通过驯化可摄食人工配合饲料。2004年笔者在河南省郑州市沟赵镇水产养殖场进行了黄颡鱼规模化养殖高产课题试验,取得了较高的经济效益,现将试验情况总结如下。1试验条件1.1池塘条件试验池塘共9口,每口池塘面积均为0.67 hm2,水深2.0~2.5 m,水源充足,水质符合渔业用水水质标准。每个池塘配备6 kW叶轮式增氧机1台。1.2池塘清整投放鱼种前约15 d以1500 kg/hm2的生石灰或375 kg/hm2的漂白粉清塘消毒,7 d后投施3000~4500 kg/hm2的有机肥料,待水体中大量出现浮游动物后投放鱼种。2试验方法2.1鱼种放养鱼种购自湖北…     

不同溶氧水平下氨氮和亚硝酸盐对黄颡鱼的急性毒性研究

在高溶氧(10.77±0.40)mg/L、中溶氧(6.89±0.33)mg/L和低溶氧(3.45±0.54)mg/L水平下,研究了氨氮和亚硝酸盐对黄颡鱼急性毒性效应。结果显示:高溶氧水平下,氨氮和亚硝酸盐对黄颡鱼的96 h-LC50值(95%可信区间)分别为148.1 mg/L(125.03～172.37 mg/L)、206.52 mg/L(164.25～246.23 mg/L);在中等溶氧水平下,氨氮和亚硝酸盐对黄颡鱼的96 h-LC50值(95%可信区间)分别为106.69 mg/L(89.92～123.70 mg/L)、145.77 mg/L(116.77～174.77 mg/L);而低溶氧水平下,氨氮和亚硝酸盐对黄颡鱼的96 h-LC50值(95%可信区间)分别为68.03 mg/L(58.32～77.89 mg/L)、81.33 mg/L(64.76～96.70 mg/L)。结果表明,在3种溶氧条件下,氨氮对黄颡鱼的毒性明显大于亚硝酸盐对黄颡鱼的毒性,因而氨氮对黄颡鱼的毒性成为其养殖过程中的重要影响因子。