中草药联用对罗非鱼无乳链球菌作用的研究

本文利用BIOLOG自动微生物鉴定系统对所分离保存的罗非鱼致病菌株进行生化鉴定,确定为无乳链球菌(Streptococcus agalactiae),进而测定虎杖、五倍子、地榆、黄连、大黄、白头翁、黄芩等14种中草药单用及联用复方对该致病菌株的抑菌效果。结果表明,从14种中草药中筛选出6种中草药(中草药A、中草药B、中草药C、中草药D、中草药E、中草药F)对无乳链球菌有良好的抑制作用;随后考察此6种中草药组合成双联用、三联用及四联用复方的抑菌效果,其中双联用中的2种复方具有显著协同作用,4种复方具有协同作用;三联用中的8种复方具有显著协同作用,3种复方具有协同作用;四连用中的1种复方具有显著协同作用,1种复方具有协同作用。     

复方中草药对尼罗罗非鱼非特异性免疫功能的影响

研究了在饲料中添加一种以鱼腥草为主要成分(占40%)的复方中草药制剂对尼罗罗非鱼(Oreochromis niloticus)非特异性免疫指标的影响。将鱼腥草、黄芩、板蓝根、连翘、金银花等10种中草药按一定比例充分粉碎后混匀制成以鱼腥草为主要成分的复方中草药制剂,按质量分数0%(对照组)、1%(A组)、3%(B组)和6%(C组)分别添加到基础饲料中饲喂尼罗罗非鱼,饲养12周。分别在第4、8和12周采集各组尼罗罗非鱼血液,测定相关非特异性免疫指标。结果显示:从第8周开始,B组尼罗罗非鱼血清丙二醛(MDA)含量显著降低,C组尼罗罗非鱼血清溶菌酶(LSZ)活力、超氧化物歧化酶(SOD)活力、一氧化氮(NO)含量显著提高,MDA含量显著降低。在第12周,B组和C组尼罗罗非鱼白细胞吞噬百分比、吞噬指数和血细胞呼吸爆发活力、血清SOD活力、LSZ活力、NO含量都显著提高,MDA含量则显著降低。结果表明:在饲料中添加3%和6%质量分数的以鱼腥草为主要成分的复方中草药制剂均能增强尼罗罗非鱼的非特异性免疫力,其中以添加比例为6%的效果最好。     

棕点石斑鱼中草药免疫增强剂的快速筛选

采用离体外周血白细胞与中草药水提液共同孵育法,快速筛选棕点石斑鱼中草药免疫增强剂。39种生药量浓度为100 mg/ml 的中草药水提液及添加5.0 mg/ml 酵母聚糖的中草药水提液分别与棕点石斑鱼外周血白细胞孵育后,采用氮蓝四唑(NBT)还原法检测各种中草药水提液对棕点石斑鱼外周血白细胞氧呼吸爆发活性的影响,再以吞噬乳胶微球法检测具有显著增强白细胞氧呼吸爆发活性效果的中草药对棕点石斑鱼白细胞吞噬活性的影响,筛选中草药免疫增强剂,并将其拌料饲喂棕点石斑鱼,考察其对棕点石斑鱼外周血和头肾白细胞氧呼吸爆发活性的影响。结果显示,39种中草药中有10种对棕点石斑鱼离体白细胞氧呼吸爆发活性显著提高15%以上,3种酵母聚糖添加组对白细胞氧呼吸爆发活性提高70%以上;筛选出可同时提高棕点石斑鱼离体白细胞的氧呼吸爆发活性和吞噬活性的 3种中草药,它们分别为鸡血藤、黄柏和墨旱莲。拌料饲喂实验结果显示,饲喂1%的鸡血藤、黄柏和墨旱莲可显著提高棕点石斑鱼体内外周血和头肾白细胞氧呼吸爆发活性。     

尼罗罗非鱼精巢支持细胞分离培养体系建立及优化

本研究分离培养尼罗罗非鱼(Oreochromis niloticus)精巢支持细胞(Sertoli cells, SCs)，建立并优化尼罗罗非鱼精巢支持细胞分离培养体系。无菌条件下，取发育至第Ⅲ期的尼罗罗非鱼精巢，PBS清洗后，剪碎精巢组织，0.25%胰蛋白酶消化，用含10%犊牛血清(Newborn bovine serum, NBS)的L-15培养液终止消化，过滤、离心，获得细胞。差速贴壁法获得SCs后，在26℃、无CO2饱和湿度恒温培养箱中培养。分别采用含10% NBS的L-15、M199、F12培养液，含5%、10%、15% NBS的L-15培养液，含1%罗非鱼血清的L-15+10% NBS培养液培养尼罗罗非鱼SCs。各组每2 d取细胞计数，绘制SCs生长曲线；甲基绿染色，倒置显微镜下观察SCs的形态。尼罗罗非鱼SCs培养1~2 d，细胞贴壁；培养3~5 d，细胞完全贴壁并迅速增殖。单个SCs呈不规则多边形，其核位于胞质中央，呈卵圆形，胞质中可见吞噬颗粒和空泡，空泡聚集于支持细胞胞质的两极或散布于核的四周。SCs在L-15培养液中贴壁生长，在F12、M199培养基中较难贴壁。与F12、M199培养液相比，L-15培养液更有助于尼罗罗非鱼SCs生长增殖(P<0.01)。与添加5%和15% NBS相比，在L-15培养基中添加10% NBS更有助于尼罗罗非鱼SCs生长增殖(P<0.05)。与未添加尼罗罗非鱼血清相比，在10% NBS+L-15培养中添加1%尼罗罗非鱼血清，能显著促进SCs生长增殖(P<0.05)。研究表明，采用胰酶消化差速贴壁法获得尼罗罗非鱼精巢支持细胞，10% NBS+1%罗非鱼血清+L-15培养液培养，能显著促进尼罗罗非鱼精巢支持细胞生长增殖。     

虎杖提取物对尼罗罗非鱼脂肪肝的干预作用

为探究虎杖提取物对尼罗罗非鱼(Oreochromis niloticus)脂肪肝的干预作用,选取120尾健康尼罗罗非鱼,随机分为4组,分别投喂基础饲料(正常组,粗脂肪6%),高脂饲料(高脂组,粗脂肪21%),以及含2.5、5 g/kg虎杖提取物的高脂饲料。饲养60 d后,采集尼罗罗非鱼血液和肝组织,并测定血脂水平、抗氧化状态和基因表达。结果显示:与正常组相比,高脂饲料投喂显著提高了尼罗罗非鱼血脂水平、引起了氧化应激和炎症反应、最终导致了肝损伤发生。与高脂组相比,虎杖提取物组明显抑制了血清血脂水平和谷丙转氨酶(GPT)活性上升,以及肝组织中超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)和谷胱甘肽(GSH)水平的下降;同时虎杖提取物显著降低了肝组织中丙二醛(MDA)和肝脂肪含量;此外,基因表达数据显示,虎杖提取物显著阻碍了白细胞介素-1β(IL-1β)的上调和过氧化物酶体增殖物激活受体α(PPAR-α)下调。结果表明虎杖提取物可明显缓解高脂饲料引起的尼罗罗非鱼脂肪性肝损伤。     

31种中草药及其复方对维氏气单胞菌体外抑菌研究

为筛选对昆明裂腹鱼源维氏气单胞菌(Aeromonas veronii)有抑菌作用的中草药及复方,本研究通过煎煮法制备31种常见中草药水提取物,以牛津杯法测定各味中草药对该菌的抑菌圈直径,采用二倍稀释法测定最小抑菌浓度(MIC)和最小杀菌浓度(MBC);选取体外抗菌活性较好的6种中草药,应用棋盘法进行15组二联药方的部分抑菌浓度指数(FIC)试验;按正交设计表配伍25组复方,同样进行药敏试验。结果显示,31种中草药中,五倍子、地榆、诃子、苏木、黄芩、黄连等6种中草药抑菌效果最好;15组联用药方均未发生拮抗作用,其中以五倍子+地榆、诃子+黄芩联用效果最好,FIC分别为0.31、0.50,表现为协同作用;综合复方中草药的抑菌作用,复方F16(五倍子∶地榆∶诃子∶苏木∶黄芩∶黄连=4∶4∶2∶5∶3∶1)的抑菌效果最佳,抑菌圈直径达23.2 mm, MIC与MBC分别为0.98 mg/mL、1.95 mg/mL,可作为昆明裂腹鱼源维氏气单胞菌抑菌药物的首选配伍组合。     

杂交鲟中草药免疫增强剂的体外快速筛选

采用杂交鲟的离体外周血和中草药的水提液进行共同孵育的方法,快速筛选杂交鲟中草药免疫增强剂。实验选用质量为365g,体长18cm的杂交鲟鱼1尾,抽取杂交鲟鱼的血样,提取其白细胞,用苦参、茵陈、杜仲、淫羊藿、茯苓、紫草、甘草、生地等八种中草药浓度为0.5g/mL的水提液与杂交鲟的外周血白细胞共同孵育后,分别采用抗超氧阴离子试剂盒、金黄色葡萄球菌法来测定杂交鲟的氧呼吸爆发活性以及中草药对杂交鲟白细胞的吞噬活性(吞噬活性以吞噬百分比(phagocytic percent-age,PP)和吞噬指数(phagocytic index,PI)表示)的影响。结果表明:各处理组外周血白细胞氧呼吸爆发活性均存在显著差异(P0.05),其中苦参、茵陈、杜仲、淫羊藿、茯苓、紫草和甘草组显著高于对照组(P0.05),生地显著低于对照组(P0.05)。苦参、茯苓、生地组外周血白细胞吞噬百分比显著高于对照组(P0.05)。苦参、生地组外周血白细胞吞噬指数显著高于对照组(P0.05),紫草和甘草组外周血白细胞吞噬指数显著低于对照组(P0.05)。结论:结合上述评价指标,苦参、茯苓更加适合作为杂交鲟候选免疫增强剂。     

不同方法灭活的痢疾志贺氏杆菌菌苗对罗非鱼免疫原性的影响

用福尔马林、苯酚以及热灭活的痢疾志贺氏杆菌作为免疫原,分别接种尼罗罗非鱼后,通过测定受免鱼的血清凝集抗体效价、溶菌酶活性、补体C3、C4以及血液白细胞的吞噬活性,用痢疾志贺氏杆菌活菌攻毒的方法,探讨3种不同方法制备的免疫原对罗非鱼免疫原性的试验结果,结果表明,3种不同方法的免疫原对罗非鱼均具有较强的免疫原性,受免鱼的血液凝集抗体效价;与对照鱼相比,白细胞的吞噬活性明显上升,受免鱼对痢疾志贺氏杆菌活菌攻毒均产生了较强的免疫保护力.     

不同盐度下3种罗非鱼的Na+-K+-ATPase活性比较

在盐度为0、10、20、30时测定了萨罗罗非鱼(Sarotherodon melanotheron)、尼罗罗非鱼(Oreochromis niloticus)和以色列红罗非鱼(Oreochromis sp.)鳃和肾中Na+-K+-ATPase活性。结果表明:(1)不同盐度对不同组织的Na+-K+-ATPase活性有显著影响,鱼的品种及其他交互作用对Na+-K+-ATPase活性均无显著影响;(2)在实验盐度范围内,无论是鳃和肾,萨罗罗非鱼、尼罗罗非鱼和以色列红罗非鱼的Na+-K+-ATPase活性均随盐度的升高而增高;3种罗非鱼中,萨罗罗非鱼的Na+-K+-ATPase活性随盐度的升高增大最为剧烈,以色列红罗非鱼次之,尼罗罗非鱼最小;低盐度时尼罗罗非鱼的Na+-K+-ATPase活性相对较高,高盐度时萨罗罗非鱼的Na+-K+-ATPase活性相对较高,Na+-K+-ATPase的活性与罗非鱼的耐盐能力有着一定的联系;(3)盐度大于7.19和11.94时,萨罗罗非鱼鳃中的Na+-K+-ATPase活性分别开始高于以色列红罗非鱼和尼罗罗非鱼;盐度大于18.42时,萨罗罗非鱼肾中的Na+-K+-ATPase活性开始高于尼罗罗非鱼;(4)除了在盐度20和30中的尼罗罗非鱼及盐度30的以色列红罗非鱼,是肾中的Na+-K+-ATPase活性高于鳃外,其他均为鳃中的Na+-K+-ATPase活性高于肾,肾中Na+-K+-ATPase活性在不同盐度之间的变化较鳃剧烈;罗非鱼的鳃比肾在渗透压调节上起的作用更大。     

梭子蟹乳化病病原菌联合药敏实验

采用棋盘微量稀释法对分离自患乳化病梭子蟹的致病菌溶藻弧菌进行中草药与化学药剂联用的体外联合药敏实验。选择五倍子、地榆、石榴皮以及复方新诺敏、磺胺＋TMP、庆大霉素作为联合用药药物，结果发现联合用药时各药物的MIC值比单独使用时有显著降低，具有统计学意义（P〈0．05），其中以五倍子和庆大霉素的协同作用最为显著。     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

中草药复合微生态制剂对吉富罗非鱼(Oreochromis niloticus)生长、肠道菌群及抗病力的影响

通过在饲料中分别添加2×107 CFU/g的芽孢杆菌制剂、中草药芽孢杆菌制剂、复合微生态制剂和中草药复合微生态制剂,研究4种微生态制剂对吉富罗非鱼(Oreochromis niloticus)生长、肠道菌群及抗病力等的影响。结果显示：(1)饲料中添加4种微生态制剂均可显著提高罗非鱼的增重率(P<0.05),对成活率和饲料利用率也有一定程度的提高(P>0.05),而中草药复合微生态制剂对罗非鱼促进生长效果最佳。(2)饲料中添加4种微生态制剂可以显著提高罗非鱼肠道中的细菌总数、芽孢杆菌、乳酸杆菌和双歧杆菌数量(P<0.05),大肠杆菌数量显著低于对照组(P<0.05),说明饲料中添加一定量的4种微生态制均可改善罗非鱼的肠道菌群结构,以中草药复合微生态制剂的改善效果最佳。(3)经人工感染无乳链球菌后,罗非鱼对照组全部死亡,4个实验组只有部分死亡。鉴定发现,吉富罗非鱼的死亡均由感染无乳链球菌所致,试验组罗非鱼的免疫保护率分别为51.42%(B组)、58.62%(C组)、58.62%(D组)和68.93%(E组),以中草药复合微生态制剂组的免疫保护率最高。综上所述,在饲料中添加一定比例的中草药复合微生态制剂可以提高吉富罗非鱼生长指标、改善其肠道菌群结构和增加抗病力。     

The feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants

An analysis of the immune activity of herbal extract in vivo and in vitro assays was performed to explore the feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants. In vitro experiment was conducted by adding herbal extract into the medium of primary cultured hemocytes and incubating for 24h. In vivo experiment was carried out by feeding shrimp with diets incorporated with herbal extract for 21 days. Challenge test can be used to directly reflect comprehensive immune capacity of organisms. Four immune indexes, total hemocyte counts (THC and lysozyme, phenoloxidase (PO), and phagocytic activity were examined both in vitro and in vivo. The results showed that effects of herbal extract on THC or lysozyme activity were different between in vitro and in vivo. On the contrary, effects of herbal extract on PO activity and phagocytic activity in vitro and in vivo showed a strong correlation. Herbal extract, which enhanced PO activity and phagocytic activity in vitro, had ultimate immune-promoting function in vivo. Therefore, it was feasible to use primary hemocyte culture to screen herbal immunostimulants by determining the effects of herbal immunostimulants on PO and phagocytic activities.     

Effects of Isatis root polysaccharide on non‐specific immune responses and nutritive indices in obscure pufferfish,Takifugu obscurus

This study examined the effects of Isatis root polysaccharide (IRPS) on the growth and non‐specific immunological responses of obscure pufferfish (Takifugu obscurus) as well as the potential of using this Traditional Chinese Medicine for disease prevention. The antioxidant capacities, lysozyme and complement C3 activities in phagocytosis, erythrocyte C3b receptor rosette rates, respiratory burst activity, body weight increase (BWI), feed conversion ratio (FCR) and several other nutritive indices were determined in pufferfish fed a control diet or diets containing either a low or high level of IRPS. The results showed that IRPS played a positive role in regulating the leukocyte phagocytic rate, respiratory burst and antioxidant enzymes. The IRPS‐fed fish showed an increased BWI and reduced FCR compared with pufferfish fed the control diet. The significant alterations of non‐specific immune response parameters in fish fed the less IPRS diet were rapid, occurring within 7 days, but were then reduced, whereas those in fish fed the more IRPS diet peaked at 10–15 days of treatment but were maintained for at least 30 days. Isatis root polysaccharide activates host immune function, improves phagocytosis by macrophages and exerts direct antimicrobial and antiviral effects, thereby helping to resist infection.     

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。     

10种中草药及4种组方对罗非鱼致病性海豚链球菌体外抑菌试验

采用琼脂扩散法(打孔法)研究了黄连等10种中草药及4种组方对尼罗罗非鱼(Oreochromis niloticus)致病性海豚链球菌(Streptococcus iniae)的抑制作用,并通过二倍稀释法测定抑菌作用较强的中草药对海豚链球菌的抑菌和杀菌浓度.结果显示:10种中草药对海豚链球菌均有不同程度的抑制作用,其中黄...     

复方中草药对奥尼罗非鱼血液非特异性免疫功能的影响

在基础饲料中添加3个不同水平的复方中草药分别饲养奥尼罗非鱼,旨在探讨复方中草药对奥尼罗非鱼血液非特异性免疫功能的影响.挑选质量(36±5)g的健康奥尼罗非鱼,共480尾,随机分为8组,每组60尾,每组设置1个平行.分别用含有0‰(对照组)、0.5‰、1.0‰、1.5‰复方中草药的饲料喂养60 d,每15 d分别取血样,...     

复方中草药对青虾免疫功能的影响

将大黄、黄芩、党参、板蓝根等十余味中草药粉碎过筛后制成复方添加剂按1%、2%、3%的比例添加于基础饲料中制成颗粒药饵投喂青虾,测定青虾相关免疫指标的变化。结果表明,青虾血细胞吞噬百分比和吞噬指数、血清溶菌酶活力均显著提高。经嗜水气单胞菌攻毒后,各试验组的免疫保护率也明显提高。在各试验组中以2%的添加量的效果最好。试验证明,该添加剂可显著地提高青虾的免疫功能,能有效地预防嗜水气单胞菌的感染。     

Role of probiotics on the immunity of Nile tilapia Oreochromis niloticus: a review

The development and intensification of Nile tilapia farming systems has happened together with ever increased use of antibiotics and therapeutants. Research on the use of probiotics has come along as alternative to routine use of antibiotics in fish diets. The manipulation of the gut microbiota through dietary probiotic inclusion targeting improved growth rate and disease resistance has been investigated in many farmed fish species. However, mechanisms underlying modulation of fish immune response remains unclear. This review summarizes and discuss recent findings in the role of probiotics on the immunity of Nile tilapia Oreochromis niloticus addressing available information regarding immunological parameters such as phagocytic activity, lysozyme, respiratory burst activity, antioxidant enzymes, complement system as well as immune-related gene expression, in the aim to foster research efforts on effective strategies for the successful use of probiotics in the tilapia farming industry.