Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40 ± 3.1% to 79.4 ± 1.7%) and it decreased from August to November (63 ± 2.3% to 30 ± 2.6%) in olive flounder farms at Jeju Island, South Korea during 2000-2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49 ± 7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50 ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100 ppm for 30 min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.     

牙鲆迟钝爱德华氏菌感染症及其病原的研究

对 7起牙鲆迟钝爱德华氏菌感染病例进行了发病情况、临床特征、病理变化等方面的检验 ,经对细菌的分离与鉴定表明所检病例均为迟钝爱德华氏菌的单独感染 ,系统归纳了该感染症的主要特点。同时 ,对所分离后做纯培养的 130株迟钝爱德华氏菌进行了主要生物学性状、血清型的测定 ,表明除在生化试验的吲哚项目中表明有株间差异 (阴性的 2 0株、阳性的 110株 )外 ,130株对其他所测内容的结果一致 ,130株均为同种血清型。从每起病例分离并鉴定的各 1个代表菌株做对健康牙鲆的人工感染试验 ,表明了相应的原发病原学意义及较强的致病作用。药敏试验结果表明 ,对供试 37种抗菌药物中的头孢唑啉等 19种药物敏感、对青霉素G等 5种药物耐药、对氨苄青霉素等 13种药物表现了株间差异。经以荧光抗体技术对纯培养物、人工感染病死鱼肝脏中细菌的检验 ,初步表明了荧光抗体技术在对迟钝爱德华氏菌检验中作为辅助检验手段的可行性。     

牙鲆Ⅰ型甲腺原氨酸脱碘酶基因片段的克隆及序列分析

在GenBank中查找到已知的斑马鱼、奥尼罗非鱼、金头鲷、爪蟾等物种的Ⅰ型甲腺原氨酸脱碘酶的基因序列并运用Primer Premier 5.0软件对这些物种的Ⅰ型甲腺原氨酸脱碘酶基因序列进行同源性分析。依据同源性较大的保守区基因序列设计一对引物,并提取变态期牙鲆仔鱼总RNA作为模板进行RT-PCR,得到一条cDNA片段。将产物连接到T载体中,转化、扩增重组质粒。提取大肠杆菌中的重组质粒进行DNA测序。将DNA测序所得到的基因序列翻译成蛋白序列和已知的奥尼罗非鱼、金头鲷、底鳉、斑马鱼的Ⅰ型甲腺原氨酸脱碘酶基因序列进行同源性分析。结果显示,利用RT-PCR的方法扩增出一条280 bp的目的基因片段,比对结果发现其和奥尼罗非鱼、金头鲷、底鳉、斑马鱼的Ⅰ型甲腺原氨酸脱碘酶基因的蛋白序列的同源性分别为85%,83%,81%,71%。对蛋白序列进行生物信息学分析发现整个蛋白序列中无信号肽,无糖基化位点,无跨膜结构域,但含有一个典型的T4_deiodinase结构。在N-端第69～70区段有β-折叠中心;第35～37区段可能形成转角或无规则卷曲;第8～13区段,第16～20区段等区段是柔性区域。D1蛋白N端第8～11区段,第47～55区段,第90～91区段为优势抗原表位区域。本实验所获得的基因序列是前尚未见报道的牙鲆Ⅰ型甲腺原氨酸脱碘酶基因序列的一部分,为进一步研究该基因的结构与生物学功能奠定了基础。     

蛋白质限制后恢复投喂对牙鲆幼鱼生长的影响

2004年4月4日~5月22日,在(23.0±0.5)℃条件下研究了牙鲆(平均体重,(8.80±0.18)g)继蛋白质限制后恢复投喂对其生长的影响。整个实验期间对照组(C组)连续48d饱食投喂含能17.29kJ/g,含粗蛋白50.00%的饵料。蛋白质限制阶段(1~18d)处理组T30和T40的饵料蛋白含量分别为27.95%和40.47%,但饵料含能与对照组相同,在恢复投喂阶段(19~48d)各处理组均投喂与对照组相同的饵料。实验结果表明,在蛋白质限制阶段,饵料蛋白质水平下降显著降低了处理组鱼的体重、特定生长率、饵料湿重、干重以及能量转化率(p<0.05),而摄食率、蛋白质效率和饵料蛋白质转化率随饵料蛋白水平降低而显著升高(p<0.05)。恢复投喂结束后,处理组上述各指标均恢复至对照组水平(p>0.05)。此外,除实验结束时T30组鱼体脂肪含量显著低于对照组(p<0.05)外,整个实验期间各处理组的表观消化率及鱼体成分与对照组相比均无显著差异(p>0.05)。实验结果表明,牙鲆幼鱼继蛋白质限制后的恢复投喂阶段出现了完全补偿生长效应。     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳检测,结果表明:1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

牙鲆出血病病原的初步研究

从患出血病的牙鲆 (Paralichthysolivaceus)的肝、脾、肾组织中分离到 3个菌株。经人工感染证实PHK致病性强 ,为病原菌 ,经鉴定为沙鱼弧菌 (Vibriocarchariae)。药敏试验结果显示 ,该菌株对红霉素、庆大霉素、先锋噻肟、菌必治、链霉素、氟嗪酸、多粘菌素B、头孢西丁、环丙沙星、头孢克肟、先锋必敏感 ;对萘啶酸、头孢噻吩、氯霉素、四环素、先锋Ⅴ、痢特灵、青霉素G、先锋孟多、氨苄青霉素、强力霉素等不敏感。     

Interactive effect between dietary valine and isoleucine on growth performance,blood parameters and resistance against low salinity stress of Japanese flounder Paralichthys olivaceus

The interaction between dietary valine (Val) and isoleucine (Ile) on growth, blood parameters, and tolerance against low salinity stress of Japanese flounder Paralichthys olivaceus was studied in a 60-day feeding trial. Fish (initial body weight: 3.57 ± 0.05 g) were fed six experimental diets containing different proportions of Ile and Val, two levels of Ile 0% (LI) and 2% diet (HI), combine with three levels of Val 0% (LV), 0.83% (MV), and 2.27% diet (HV), respectively. The results revealed that significant interactive effect of Ile and Val was found on body weight gain and feed conversion ratio, also stimulative effect was found in high Ile groups. Meanwhile, interactive effect of these two amino acids was also observed on high-density lipoprotein (HDL), the maximum of Ile with maximum of Val group has the highest concentration of HDL, which was significantly higher than other groups (p < .05). Moreover, no interaction was found on whole body proximate composition, muscle Ile and Val concentration, and tolerance ability against low salinity stress (reflected by LT₅₀). But high level of Ile combined with high level of Val diet altered tolerance ability against low salinity stress of Japanese flounder. It would be indicated that dietary Ile and Val interacted in the diet of Japanese flounder, and the flounders fed the diet supplemented with 2% Ile and 2.27% Val was superior to the other groups on growth and blood parameters, and tolerance against low salinity stress.     

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and Müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.     

Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus)

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis , respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges–Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.