【作者】 唐毅；

【导师】 万永继；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2008， 硕士

【摘要】 草鱼、白鲢作为我国传统的优良淡水养殖品种,一直深受广大渔农欢迎。尤其是近年来,市场多样化的需求使得草鱼、白鲢的需求量逐年递增,价格稳中有升,加上青饲料养殖草鱼成本低,效益高,白鲢摄食水体中的天然饵料生物,符合人们对天然食品的需求。因此,草鱼、白鲢如今更是被广泛推广养殖。但由于随着水产集约化养殖的发展,各种养殖方式的放养密度越来越大,人工配合饲料投喂量大大增加,养殖水体的水质也日益恶化。加上人们对防病治病的意识落后,水产病害研究滞后,因而多种细菌性疾病如细菌性烂鳃病、赤皮病和肠炎病等,白鲢细菌性败血症、烂鳃病等传染性疾病的危害更加严重,已成为严重影响其健康养殖和制约其产量与经济效益提高的主要因素,被称为阻碍草鱼、白鲢养殖规模化发展的瓶颈。本论文针对草鱼、白鲢所患几种严重的细菌性疾病,进行了致病菌的分离、鉴定及其有关特性的测定,并进行了常用抗生素和中草药对草鱼细菌性烂鳃、肠炎、赤皮病病原菌和白鲢肺炎克雷伯氏菌的药物敏感性试验,研究比较了采用福尔马林灭活荧光假单胞菌所制成的浸泡疫苗、注射疫苗和口服微囊疫苗对健康草鱼种的免疫效果。1草鱼细菌性烂鳃、肠炎、赤皮病致病菌的分离及其特性鉴定分别从患烂鳃病、肠炎病和赤皮病的草鱼体上分离出2株（CLS-01、CLS-02）、1株（CCY-01）和2株（CCP-01、CCP-02）纯化细菌。经形态学、染色特性、溶血性和理化特性鉴定,分别为柱状屈扰杆菌（Flexibacter columnaris）、肠型点状气单胞菌（Aeromonaspunctata f.intestinalis）和荧光假单胞菌（Pseudo-monasfluorescens）。致病性试验结果表明,3种分离菌使试验草鱼分别于96h、48h和96h内的死亡率达到100%、95%和100%,并从病死草鱼体中回收到与自然病例相同的分离菌。中西药药敏试验表明,3种分离菌除对氨苄西林钠、苦参、马鞭草、柴胡、穿心莲、大青叶、中草药复方3、复方6完全不敏感（CCP-01对黄柏、黄连、地锦草和筋骨草,CLS-01对筋骨草、鱼腥草亦不敏感）以外,对其它试验用抗生素和中草药及其复方均呈现不同程度的敏感性。其中CLS-01对氧氟沙星（50mg/L）、CCY-01对恩诺沙星（50 mg/L）利诺氟沙星（50mg/L）以及3种分离菌对五倍子、中草药复方1、复方2、复方5、复方9均极度敏感。恩诺沙星、氟苯尼考、五倍子和黄芩对3种菌（CLS-01、CCY-01、CCP-01）的最小抑菌浓度分别为（0.391mg/L、0.049mg/L、0.781mg/L）,（0.977mg/L、0.4883mg/L、31.250mg/L）,（0.781mg/mL、1.563mg/mL、0.391mg/mL）,（1.563mg/mL、6.250mg/mL、25.000mg/mL）:最小杀菌浓度分别为（1.562mg/L、0.195mg/L、3.125mg/L）,（3.906mg/L、3.9062mg/L、125.000mg/L）,（3.125mg/mL、6.25 mg/mL、1.563mg/mL）,（15.625mg/mL、25.000mg/mL、100.000mg/mL）。结果显示,恩诺沙星、氟苯尼考、五倍子和黄芩对以上三种水产致病菌均表现出较强的抑菌和杀菌作用。在安全浓度范围内合理用药,安全有效。2白鲢烂鳃病致病菌的分离鉴定及其特性测定本研究从患病白鲢鳃、肝胰脏分离到致病细菌,该菌为革兰氏阴性菌,具荚膜,粗短杆状,大小约0.3-0.4×0.5-0.81μm,单个存在或两个相连:发酵葡萄糖产酸产气,乳糖、麦芽糖、甘露醇、蔗糖、枸橼酸盐、尿素显阳性;对鸟氨酸、蛋白胨水、卫茅醇、苯丙氨酸、H₂S均为阴性;V-P试验强阳性,细菌学鉴定该菌为肺炎克雷伯氏菌（Klebsiella pneumoniae）。分离菌经胸鳍基注射回归感染试验均出现与自然发病相似的症状。采用纸片扩散法和二倍稀释法分别测定了常用抗菌西药对该菌的敏感度与最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,同时采用打孔法测定了常用中草药对该菌的敏感度。结果表明:所选17种抗菌西药对分离菌（KPCQC01）均有不同程度的抑制作用,其中氧氟沙星（50mg/L）、盐酸环丙沙星（50mg/l）、诺氟沙星（50mg/l）、SMZ:TMZ（100mg/l）、氟苯尼考（50mg/l）、对该菌极度敏感,但诺氟沙星、盐酸环丙沙星和氟苯尼考的抗菌活性不如氧氟沙星和复方SMZ:TMZ强;甲氧苄定TMZ（100mg/l）、氨苄西林钠（0.2mg/l）高度敏感:复方新诺明（100mg/l）、硫酸庆大霉素（0.04万IU/ml）、甲砜霉素（50mg/l）、氯霉素（50mg/L）中度敏感;两种氯制剂、磺胺甲恶唑（100mg/l）、麦迪霉素（50mg/l）、土霉素（50mg/l）、罗红霉素（50mg/l）抑菌效果不明显;氧氟沙星和复方SMZ:TMZ2种药物,在48h内对该菌抑菌的持续性较好,且最小抑菌浓度和最小杀菌浓度分别为0.5mg/L和1.0mg/L与6.25mg/L和400mg/L。所选12种常用单方中草药中。五倍子对分离菌（KPCQC01）极度敏感;金银花、鱼腥草、黄芩和地渝高度敏感;细菌对黄连、地锦草、龙胆草等不敏感;11组中草药复方对该菌都具有较为明显的抑菌效果,其中以复方9的抑菌效果最佳,其抑菌圈直径达29.7mm。3草鱼赤皮病的免疫效应研究采用0.5%福尔马林灭活草鱼赤皮病病原菌分别制成浸泡疫苗、注射疫苗和口服微囊疫苗,然后以相应的疫苗分别浸泡（含菌数为1.5×10⁷cfu/ml的菌苗溶液）3～5cm当年夏花草鱼、0.2ml/尾注射（含菌数为1.5×10⁸cfu/ml的菌苗）10g左右的草鱼、16g/组·次微囊疫苗混合饲料（含2%微囊疫苗）投喂10g左右的草鱼。接种免疫后,分别于第21d、35d、49d、63d及77d,通过检测实验对象的血清和采用活菌攻毒的方法,探讨了灭活菌苗对预防草鱼赤皮病的效果。结果显示,所制疫苗3种免疫途径都有免疫效果。全菌灭活疫苗浸泡免疫、注射免疫和微囊化疫苗口服免疫的抗体滴度（凝集抗体效价）平均分别为1:19、1:190、1:62;用荧光假单胞菌活菌攻毒显示,3种免疫途径均产生良好的保护效应,其免疫保护率分别达41.6%和40.0%、83.4%和80%、58.4%和53.3%。更多还原

【Abstract】 Grass carp,silver carp,as China’s traditional fine varieties of freshwater aquaculture,has been welcomed by the overwhelming majority of the fishmen and consumers.Especially in recent years, the demand for market diversification makes grass carp,silver carp demand increase every year, prices rose steadily,and the Green Grass carp feed breeding’s low cost,high efficiency,silver carp’s feeding on the natural food organisms in water,both well satisfied people’s needs on natural food. Therefore,grass carp,silver carp now are more and more widely cultured.However,as with the development of aquaculture intensive cultivation,the farming methods of increasing stocking density,artificial diets fed significantly increased,the quality of aquaculture water also has been deteriorating.With the people’s awareness of preventing and curing diseases backward,aquatic disease research lags behind,thus a variety of bacterial diseases,such as bacterial gill-rot disease, skin disease and Chek enteritis disease.etc.They have seriously affected the healthy culture and constrainted the increase of its yield and fishemen’s incomes,which also becomed a bottleneck which impedes grass carp,silver carp scale aquaculture developmentThis paper researched grass carp,silver carp’s several serious bacterial disease,made separation, identification and the determination of the characteristics of the pathogens,and the sensitivity tests of commonly used antibiotics and Chinese herbal medicine on the grass carp bacterial gill-rot, enteritis,Chek Paper Pathogen and silver carp Klebsiella pneumoniae drug,the study also compared the effects of the use of formalin-inactivated Pseudomonas fluorescens manufactured by immersion vaccine,vaccination and oral health microencapsulated vaccine to species of grass carp.1.Characteristic identification and separation of grass carp’s bacterial gill-rot,enteritis,and red skin disease pathogensIsolated respectively 2(CLS-01,CLS-02),1(CCY-01)and 2(CCP-01,CCP-02)purification bacteria from grass carps which suffered from gill-rot disease,enteritis disease and red skin disease. After the morphology,staining characteristics,hemolytic and physicochemical identification,these bacteria were respectively columnar flexor nuisance bacilli(Flexibacter columnaris),intestinal point Aeromonas(Aeromonaspunctata fintestinalis)and Pseudomonas fluorescens (Pseudo-monasfluorescens).Rsult of the pathogenic test showed the 3 isolated bacteria made separately the mortality rate of grass carp reach 100%,95%,100%in 96h,48h,and 96h,and the isolated bacteria recycled from dead grass carp were same as natural bodies.The sensitivity test of Chinese and western medicine showed that in addition to ampicillin sodium,Kushen,Verbenaceae, Bupleurum,Andrographis,Daqingye,herbs Compound 3,not sensitive compound 6(CCP-01 Mr Wong,berberine,and Kam grass and skeletal grass,CLS-01 on the bones and tendons grass,not sensitive Houttuynia cordata),the 3 isolated bacteria showed different degrees of sensitivity to other antibiotics and Chinese herbal compound medicine.Of them CLS-1 to ofloxacin(50mg/L),CCY-01 to enrofloxacin(50mg/L)and norfloxacin(50mg/L)were extremely sensitive,as well as the 3 isolated bacteria to gallnut,Chinese herbal compoundl,compound 2,compound 5,Compound 9. The minimum inhibitory concentration of nrofloxacin,florfenicol,gallnut and baicalin to three kinds of bacteria(CLS-01,CCY-01,CCP-01)were respectively(0.391mg / L,0.049mg/L,78.1mg/L), (0.977mg/L,0.4883mg/L,31.250mg/L),(0.781mg/mL,1.563mg/mL,0.391mg/mL),the minimal bactericidal concentration were respectively(1.563mg/mL,6.250mg/mL,25000mg/mL);(1.562mg/L, 0.195mg/L,3.125mg/L),(3.906mg/L,3.9062mg/L,125.000mg/L),(3.125mg/mL,6.25mg/mL, 1.563mg/mL),(15.625mg/mL,25.000mg/mL,100.000mg/mL).The results showed that enrofloxacin,florfenicol,gallnut and baicalin had a strong antibacterial and germicidal effect on the 3 aquatic pathogens above.The drug would be safe and effective if used in a proper concentration.2 Characteristic identification and separation of Silver carp’s gill-rot disease pathogensThis research separated pathogen from the disease silver carp’s gill and liver.The pathogen is a gram-negative bacteria,a capsule,Cuduan rod,the size of about 0.3-0.4×0.5-0.8μm,single-existent or two adjacent:fermentation of glucose produced gas and acid,positive for lactose,maltose, mannitol,sucrose,citric acid salt,urea;negative for ornithine,peptone water,alcohol Mao Wei, phenylalanine,H2S and strong positive for VP test,it turned out to be Klebsiella pneumoniae after bacterium identification.The regression tests in which separated bacteria were injected in the bottom of pectoral fin were all presented with symptoms similar to the natural incidence.Disk diffusion and double dilution method were used to test the sensitivity of the commonly used antibody western medicine to this bacterium and to determine the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC);the sensitivity of the commonly used Chinese medicine to this bacterium was tested by the drilling method.The results show that:the selected 17 kinds of antibody western medicine all had different levels of inhibition on the isolated bacterium (KPCQC01),of them ofloxacin(50mg/L),ciprofloxacin hydrochloride(50mg/l),Connaught sparfloxacin Star(50mg/l),SMZ:TMZ(100mg/L),florfenicol(50mg/L)were extremely sensitive to this bacterium,but norfloxacin,ciprofloxacin hydrochloride and florfenic’s antibacterial activity were weaker than ofloxacin and compound SMZ;trimethoprim(100mg/L),ampicillin sodium (0.2mg/L)were highly sensitive;cotrimoxazole(100mg/L),gentamicin sulfate(00,400IU/ml), Thiamphenicol(50mg/L),chloramphenicol(50mg/L)moderately sensitive;2 chlorine preparations, sulfamethoxazole(100 mg/l),Midecamycin(50mg/L),oxytetracycline(50 mg/L),roxithromycin (50mg/L)inhibition effect was not significant;the continual effect of ofloxacin and compound SMZ: TMZ2 drugs’ inhibition to this bacterium were better in 48h,and the minimum inhibitory concentration and the minimum bactericidal concentrations were respectively 0.5mg/L and 1.0mg/L and 6.25mg/L and 400mg/L.Of the Selected 12 kinds of Chinese herb medicine,gallnut was extremely sensitive to the isolated bacterium(KPCQC01);while honeysuckle,Houttuynia cordata, and Yu baicalin were highly sensitive;the bacterium was not sensitive to Huanglian,Kam grass and gentian;11 groups of the Chinese herbal compound medicine had obvious inhibition effect on this bacterium,of which compound 9 was the best,whose diameter of the inhibition zone was 29.7mm.3 check the immune effects of grass carp in red skin diseaseInactivated grass carp’s red skin pathogen by 0.5%formalin,then made separately into immersion vaccine,vaccination and oral microencapsulated vaccine,and thenused the corresponding vaccine to soak(a few bacteria 1.5×10~7 cfu/ml the vaccine solution)3 to 5 cm fry grass carp,inject 0.2ml/tail(for the number of bacteria 1.5×10~8 cfu/ml the vaccine)to10 g-grass carp and to feed 16g/ Group·every time microencapsulated vaccine in the mixed diet(containing 2%microencapsulated vaccine)to 10g- grass carp.After immunization,analyzed the prevent results of grass carp’s red skin disease by detecting the experiment carp’serum and adopting the use of poison attack poison method in the first21 d,35d,49d,63d,77d.The results showed that all the 3 three immune system channels were effective.The average antibody titers(agglutination titer)of the Whole-cell vaccine immunization,injection immunization and microencapsulated vaccine oral immunization were respectively 1:19,1:190,1:62;the Pseudomonas fluorescens infection showed that 3 kinds of immune paths all had good protection effects,and the immune protection rates reached respectively 41.6% and 40.0%,83.4%and 80%,58.4%and 53.3%.更多还原