罗非鱼链球菌的分离鉴定及中草药药敏试验

为了对某养殖户发病的罗非鱼进行治疗,试验进行了病原菌的分离培养、生化鉴定及中药药敏试验。结果表明:从患病罗非鱼体内分离得到一株链状排列的革兰氏阳性球菌,经生理生化鉴定为无乳链球菌,回归试验证明该菌对罗非鱼有较强的致病性。药敏试验结果显示,该菌对苏木、石榴皮、五倍子、黄连、桉树叶、沙棘和乌梅高度敏感,对大黄、黄柏中度敏感。     

奶牛乳房炎无乳链球菌的分离及PCR鉴定

利用THB(Todd-Hewitt Broth)固体培养基和色素试验培养基选择培养无乳链球菌,参照GenBank中无乳链球菌参考菌株(Accession:AF015927.1、JQ289582.1)16SrRNA和种属特异性基因cfb(CAMP因子)序列设计引物,对奶样中分离的12株疑似无乳链球菌进行鉴定。结果显示,经PCR扩增后,被检测的12株细菌均可扩增出预期大小的16S rRNA基因序列,而12株中有8株可以扩增出预期大小的cfb基因序列,条带单一,特异性好。序列BLAST显示,12株菌的16S rRNA基因序列与NCBI上报道的无乳链球菌相应序列高度同源(>99.0%),各分离菌株间的16S rRNA基因序列也高度同源(99.0%～100.0%);cfb基因序列与NCBI上已报道的无乳链球菌相应序列具有高度同源性(>99.0%),各菌株间cfb基因序列也高度同源(100.0%)。经选择培养与PCR鉴定结果可以确定12株疑似菌株中有8株为无乳链球菌。     

不同剂量三黄连散对罗非鱼感染无乳链球菌防治效果的试验研究

试验旨在从临床症状、疗效评价、死亡率和保护率等方面探索三黄连散对罗非鱼感染无乳链球菌预防效果的量效关系并据此进行剂量筛选。将210尾罗非鱼随机分为7组（即1.67 g/kg剂量组、3.30 g/kg剂量组、6.60 g/kg剂量组、13.20 g/kg剂量组、26.40 g/kg剂量组、无乳链球菌感染对照组和空白对照组）,每组30尾鱼。各剂量试验组饲喂含不同剂量三黄连散的饲料,无乳链球菌感染对照组和空白对照组投喂基础饲料,并于第8天,各剂量试验组和感染组腹腔注射浓度为5.40×107CFU/mL的无乳链球菌菌液,空白对照组腹腔注射灭菌生理盐水。结果表明：攻毒后12 h,感染对照组和剂量组都出现了明显的临床症状并伴随急性死亡。感染对照组的临床症状始终保持较高分值,不同剂量药物组评分均低于感染对照组,13.20 g/kg剂量组和26.40 g/kg剂量组评分呈现出平稳且下降的趋势。无乳链球菌感染对照组的罗非鱼死亡率为73.33%,1.67 g/kg剂量组、3.30 g/kg剂量组和6.60 g/kg剂量组死亡率与感染对照组比较差异不显著（P>0.05）;而13.20 g/kg和26....     

罗非鱼无乳链球菌gap基因的原核表达及免疫原性研究

试验旨在探究罗非鱼无乳链球菌(GBS)膜蛋白gap的免疫原性。试验提取GBS的DNA,利用PCR扩增出gap基因,并与表达载体pET-28a-SUMO构建重组质粒,转化至大肠杆菌BL21;经IPTG诱导、SDS-PAGE分析和Western blot检测确定其表达效果。采用纯化的gap蛋白与弗氏佐剂乳化腹腔注射免疫罗非鱼,以间接ELISA法检测血清效价并使用GBS进行攻毒试验。结果显示,PCR扩增出1 011 bp大小的单条带gap基因,与预期一致;重组载体pET-28a-gap在23℃、0.4 mmol/L IPTG、12 h条件下成功表达49.8 ku的可溶性蛋白;ELISA检测显示,重组蛋白gap能够产生较高的血清抗体效价水平,对GBS攻毒后的罗非鱼相对保护率为64.1%。研究表明,重组蛋白gap具有较好的免疫原性,为鱼类的链球菌病的预防提供新的理论依据。     

基于代谢组学的黄芩水提物防治罗非鱼无乳链球菌病作用机制研究

为了探讨黄芩水提物防治罗非鱼无乳链球菌病的作用机制,试验将104尾体重60 g左右的吉富罗非鱼随机分为4组,分别为空白对照组、阴性对照组、防治组和模型组,每组26尾。阴性对照组和防治组均饲喂含5%黄芩水提物的日粮,空白对照组和模型组饲喂基础日粮。于饲喂后第8天进行攻毒试验,空白对照组和阴性对照组腹腔注射灭菌生理盐水,防治组和模型组腹腔注射浓度为1.5×10⁹ cfu/mL的无乳链球菌菌液,0.3 mL/尾,观察10 d,试验过程中记录各组临床症状及死亡情况,对濒死的试验鱼进行尾静脉采血,并对死亡试验鱼进行剖检,攻毒试验结束后计算各组的死亡率及5%黄芩水提物对防治组的保护率,并对其他试验鱼进行尾静脉采血。对各组血液进行液相色谱质谱(LC-MS)分析,通过构建主成分分析(PCA)模型和偏最小二乘判别分析(PLS-DA)模型筛选差异代谢物,导入京都基因和基因组百科全书(KEGG)数据库后进行通路富集分析。结果表明：攻毒后,空白对照组和阴性对照组食欲佳,未出现异常症状,死亡率均为0,剖检未见明显异常。模型组行动迟缓,食欲减退,体表破溃出血,眼球突出,机体失去平衡,出现侧游...     

宝石鲈无乳链球菌的分离鉴定及耐药性分析

2013年高温季节在广东广州2个养殖场患病宝石鲈(Scortum barcoo)病灶处共分离到2株细菌Py1和Py2。宝石鲈攻毒试验结果显示2株分离菌均具有较强毒力(半致死量分别为7.42×10⁶和1.72×10⁶ CFU/mL),且都对罗非鱼具有高度感染性(半致死量分别为3.39×10⁶和8.66×10⁵ CFU/mL)。经ATB生化鉴定及cfb基因序列分析,确定分离株为无乳链球菌(Streptococcus agalactiae),并对其进行了相关药敏试验检测,以期为该病的预防提供参考。     

俄罗斯鲟源停乳链球菌停乳亚种分离、鉴定及药敏特性研究

2016年6月~8月,湖北鲟养殖场的俄罗斯鲟发生一种以上下嘴唇充血肿胀为特征的病害。为确定其发病原因,本研究对自然发病鱼的病灶及内脏器官进行了病原菌的分离,并对分离株进行人工感染、毒力检测、生理生化特性测定及16S r RNA基因序列分析。结果从患病俄罗斯鲟体内分离到一株优势菌,人工感染试验出现与自然发病类似症状,确定其为俄罗斯鲟本次发病的病原菌,其对俄罗斯鲟幼鱼的半数致死剂量为3.64×10~4 cfu/g,该分离株生理生化特性与停乳链球菌停乳亚种一致;经16S rRNA基因序列比对及构建系统发育树结果显示,该分离株与停乳链球菌停乳亚种聚为一支,同源性在99%以上。该分离株鉴定为停乳链球菌停乳亚种(Streptococcus dysgalactiae subsp.dysgalactiae),命名为HD03。药敏试验结果表明,HD03株对环丙沙星、痢特灵及氟苯尼考等8种抗生素敏感,对苯唑西林、头孢唑林及阿莫西林等7种抗生素耐药。本研究首次证实停乳链球菌停乳亚种是俄罗斯鲟的病原菌,养殖时可以选用氟苯尼考进行防控。     

中草药防治罗非鱼无乳链球菌病的研究进展

无乳链球菌是一种广泛分布于自然界的革兰氏阳性,人畜鱼共患病原菌.近年罗非鱼链球菌病的报道越来越多,中国作为世界最大的罗非鱼养殖国,链球菌病已严重危害中国罗非鱼养殖业的健康发展.文章对无乳链球菌病原分类地位、流行病学、致病机理、常规防治方法及中草药防治罗非鱼无乳链球菌病的研究现状作一综述,以期为今后开展罗非鱼无乳链球菌病防控研究提供参考.     

奶牛乳腺炎无乳链球菌的分离鉴定

无乳链球菌是比较常见的导致奶牛乳腺炎的病原菌,该菌也是山羊、绵羊慢性乳房炎的病原菌之一,也能引起婴儿败血症、脑膜炎和肺炎等。人医临床上对该菌以B群链球菌相称。试验从内蒙古呼和浩特市周边5个牛场中患有乳房炎的病牛中采集120份乳样,通过分菌培养、形态学观察、生化试验,鉴定出14株无乳链球菌,同时对这14株菌进行了药物敏感试验、动物致病性试验等。试验结果发现,所分离到的无乳链球菌对青霉素类、氨基糖苷类药物高度敏感,对磺胺类、氟哌酸、喹诺酮类药物中度敏感。动物致病性试验对小鼠的致死率达到80%以上。     

裸花紫珠颗粒对罗非鱼源无乳链球菌体外抑菌活性研究

无乳链球菌是导致罗非鱼链球菌病的主要病原菌。为探讨裸花紫珠颗粒对无乳链球菌体外抑菌活性，采用肉汤二倍稀释法测定裸花紫珠颗粒对无乳链球菌的最低抑菌浓度(MIC)和最小杀菌浓度(MBC),同时进行标准无乳链球菌和临床分离无乳链球菌对常用抗菌药的敏感性试验。结果表明，裸花紫珠颗粒对罗非鱼源无乳链球菌的最低抑菌浓度(MIC)为10.000 mg/mL,最小杀菌浓度(MBC)为20.000 mg/mL;药敏试验结果显示临床分离株无乳链球菌对四环素、环丙沙星、氟苯尼考的敏感性降低。该结果为下一步开展裸花紫珠对罗非鱼临床保护试验研究奠定了基础。     

胰岛素受体-1在尼罗罗非鱼不同组织中的表达及其对注射葡萄糖的响应

本试验旨在研究胰岛素受体-1(IR-1)在尼罗罗非鱼不同组织中的表达及其对注射葡萄糖的响应。利用PCR扩增的方法从尼罗罗非鱼肌肉中克隆IR-1的c DNA片段,并通过半定量PCR检测,比较IR-1在肌肉、肝脏和心脏中的表达差异。选取体重约为100 g的尼罗罗非鱼160尾,随机分2组,每组4个重复,每个重复20尾。试验组腹腔注射葡萄糖(每100 g体重30 mg),对照组以相同剂量腹腔注射0.7%的无菌生理盐水。在注射前(0 h)和注射后的1、3、6和12 h分别进行采样,测定血浆葡萄糖和胰岛素含量,并通过实时荧光定量PCR检测IR-1在肌肉、心脏和肝脏中的mRNA相对表达量。结果显示:1)克隆出的IR-1 c DNA片段,其Gen Bank登陆号为JN967750,大小为1 979 bp,编码548个氨基酸。序列分析发现,尼罗罗非鱼的IR-1与其他物种比较具有很高的保守性,并具有丰富的酪氨酸激酶特征性序列。2)IR-1在尼罗罗非鱼肌肉、心脏和肝脏中均有较高的表达量,其中在肝脏和肌肉中的表达量基本一致,而在心脏中的表达量相对较低。3)试验组血浆葡萄糖含量在注射葡萄糖1 h后达到最高,并显著高于对照组(P0.05),而后开始下降,3 h后恢复到正常水平;试验组血浆胰岛素含量在葡萄糖注射3 h后达到最高,并显著高于对照组(P0.05),而后开始下降,12 h后恢复到正常水平。试验组肌肉和肝脏中IR-1 mRNA的相对表达量在注射葡萄糖后6 h时达到最高,显著高于对照组(P0.05),在12 h时恢复到正常水平;试验组心脏中IR-1 mRNA的相对表达量在注射葡萄糖后的12 h内没有发生显著变化(P0.05)。结果表明,注射葡萄糖后即刻升高了尼罗罗非鱼的血浆葡萄糖含量,相对于血浆葡萄糖含量的升高,血浆胰岛素含量的升高相对延迟,而肌肉和肝脏中IR-1 mRNA相对表达量的提高又延迟于血浆胰岛素含量的升高,从而加重了尼罗罗非鱼对葡萄糖的代谢负担。     

Sulfadimethoxine and ormetoprim residues in three species of fish after oral dosing in feed

Nile tilapia Oreochromis niloticus, summer flounder Paralichthys dentatus, and walleyes Sander vitreus were treated with Romet-30 (PHARMAQ AS, Oslo, Norway) via a medicated ration at 50 mg Romet-30 kg fish body weight(- 1) d(-1) for 10 d to compare the elimination kinetics of the test substance. This study was part of a larger effort to develop a species grouping concept for the labeling of therapeutic compounds for cultured fishes. The fish tests were conducted at the ideal water temperature for each species and at 5 degrees C lower than the ideal temperature except for summer flounder, which would not feed at the lower temperature of 15 degrees C. Test temperatures were 30 degrees C and 25 degrees C for Nile tilapia, 20 degrees C and 17 degrees C for summer flounder, and 25 degrees C and 20 degrees C for walleyes. Neither component of Romet-30 (sulfadimethoxine and ormetoprim) could be detected in samples of the edible portion of walleyes (muscle plus skin) collected at day 10 posttreatment or thereafter. In studies with summer flounder, only one fish had a detectable concentration of either component on day 21 or thereafter. Elimination of Romet-30 by Nile tilapia was extremely rapid. The limited number of Nile tilapia with detectable sulfadimethoxine or ormetoprim during the posttreatment period prevented the determination of elimination half-life or elimination in this species.     

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (p<0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.     

Adjuvant and immunostimulatory effects of beta-glucan administration in combination with lipopolysaccharide enhances survival and some immune parameters in carp challenged with Aeromonas hydrophila

Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses.     

Depletion study and estimation of the withdrawal period for oxytetracycline in tilapia cultured in Brazil

Defining pharmacokinetic parameters and depletion intervals for antimicrobials used in fish represents important guidelines for future regulation by Brazilian agencies of the use of these substances in fish farming. This article presents a depletion study for oxytetracycline (OTC) in tilapias (Orechromis niloticus) farmed under tropical conditions during the winter season. High performance liquid chromatography, with fluorescence detection for the quantitation of OTC in tilapia fillets and medicated feed, was developed and validated. The depletion study with fish was carried out under monitored environmental conditions. OTC was administered in the feed for five consecutive days at daily dosages of 80 mg/kg body weight. Groups of ten fish were slaughtered at 1, 2, 3, 4, 5, 8, 10, 15, 20, and 25 days after medication. After the 8th day posttreatment, OTC concentrations in the tilapia fillets were below the limit of quantitation (13 ng/g) of the method. Linear regression of the mathematical model of data analysis presented a coefficient of 0.9962. The elimination half-life for OTC in tilapia fillet and the withdrawal period were 1.65 and 6 days, respectively, considering a percentile of 99% with 95% of confidence and a maximum residue limit of 100 ng/g. Even though the study was carried out in the winter under practical conditions where water temperature varied, the results obtained are similar to others from studies conducted under controlled temperature.     

Pharmacokinetic study of enrofloxacin in Nile tilapia (Oreochromis niloticus) after a single oral administration in medicated feed

The objective of this study was to evaluate the disposition kinetics of enrofloxacin (ENR) in the plasma and its distribution in the muscle tissue of Nile tilapia (Oreochromis niloticus) after a single oral dose of 10 mg/kg body weight via medicated feed. The fish were kept at a temperature between 28 and 30 °C. The collection period was between 30 min and 120 h after administration of the drug. The samples were analyzed by high‐performance liquid chromatography with a fluorescence detector (HPLC‐FLD). The ENR was slowly absorbed and eliminated from the plasma (C_max = 1.24 ± 0.37 μg/mL; T_max = 8 h; T_1/2Ke = 19.36 h). ENR was efficiently distributed in the muscle tissue and reached maximum values (2.17 ± 0.74 μg/g) after 8 h. Its metabolite, ciprofloxacin (CIP), was detected and quantified in the plasma (0.004 ± 0.005 μg/mL) and muscle (0.01 ± 0.011 μg/g) for up to 48 h. After oral administration, the mean concentration of ENR in the plasma was well above the minimum inhibitory concentrations (MIC₅₀) for most bacteria already isolated from fish except for Streptococcus spp. This way the dose used in this study allowed for concentrations in the blood to treat the diseases of tilapia.     

Dietary Supplementation with Allspice Pimenta dioica Reduces the Occurrence of Streptococcal Disease during First Feeding of Mozambique Tilapia Fry

Abstract

Allspice Pimenta dioica as a feed additive was studied for its effects on growth performance and disease resistance in Mozambique Tilapia Oreochromis mossambicus. Five isonitrogenous (36% crude protein) and isocaloric (18.5 kJ/g) diets were formulated to contain 0 (control), 5, 10, 15, or 20 g of allspice/kg of fish feed. In a 50-d feeding trial, 15 plastic tanks (21 L) were stocked with 35 fish fry (0.012 g) each. After the feeding trial, fish were exposed to Streptococcus iniae and mortalities were recorded. The second-order polynomial regression indicated that the dietary allspice level of 10 g/kg provided the best growth performance and feed utilization. The greatest survival after pathogen challenge was also obtained from the diet supplemented with allspice at 10 g/kg. Therefore, allspice acts as a growth promoter to improve feed utilization and weight gain in Mozambique Tilapia fry and acts an antimicrobial agent to enhance disease resistance during first feeding of fry. These results suggest that allspice can be used as an alternative to antibiotics in controlling streptococcal disease in tilapia culture.

Received October 19, 2012; accepted January 20, 2014     

Effects of well-boat transportation on the muscle pH and onset of rigor mortis in Atlantic salmon

During the transport of salmon (Salmo salar), in a well-boat, 10 fish were sampled at each of six stages: in cages after crowding at the farm (stage 1), in the well-boat after loading (stage 2), in the well-boat after eight hours transport and before unloading (stage 3), in the resting cages immediately after finishing unloading (stage 4), after 24 hours resting in cages, (stage 5) and in the processing plant after pumping from the resting cages (stage 6). The water in the well-boat was at ambient temperature with recirculation to the sea. At each stage the fish were stunned percussively and bled by gill cutting. Immediately after death, and then every three hours for 18 hours, the muscle pH and rigor index of the fish were measured. At successive stages the initial muscle pH of the fish decreased, except for a slight gain in stage 5, after they had been rested for 24 hours. The lowest initial muscle pH was observed at stage 6. The fishes' rigor index showed that rigor developed more quickly at each successive stage, except for a slight decrease in rate at stage 5, attributable to the recovery of muscle reserves.     

七种常用渔药及组合对罗非鱼致病性海豚链球菌的体外抗菌试验

探讨了7种常用抗菌渔药对罗非鱼致病性海豚链球菌的体内外抗菌作用。采用二倍稀释法测定了7种渔药对致病菌的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）;在初筛基础上以MIC为单位,1×MIC、5×MIC、10×MIC和20×MIC下平板抑菌圈直径为参数,分别比较所筛选5种药物抗菌效果并优化最佳参数;利用优化的最佳参数比较5种药物单独用药与联合用药对海豚链球菌的抑菌效果;并通过拌饵投喂给药方式对罗非鱼进行5种药物治疗性给药试验。结果表明,抗菌先锋对海豚链球菌的MIC和MBC最小,分别为6.4、12.8μg/mL,其次是伯乐立康,均为12.8μg/mL;而鱼康和肠炎烂鳃灵即使在1638.4μg/mL下也无抗菌效果。5倍MIC下抑菌圈直径为本试验筛选抗菌药物的最佳参数。5 MIC时,海豚链球菌对伯乐立康和抗菌先锋敏感,对菌必清、菌毒康和海鱼安中度敏感。菌毒康与海鱼安的联合抗菌效果最佳。鱼体内抗菌试验结果表明,抗菌先锋和伯乐立康的治疗有效率超过75%,达到了理想的治疗效果,是防治罗非鱼海豚链球菌病的首选药物。