拮抗菌对病原性溶藻弧菌(Vibrio alginolyticus)粘附大黄鱼(Pseudosciaena crocea)表皮粘液的影响

采用3H-TdR同位素示踪法研究了病原性溶藻弧菌和9株拮抗菌对大黄鱼表皮粘液的粘附动力学。结果表明,10株细菌的粘附作用都符合饱和粘附动力学特征;R4、R13和R32等3株拮抗菌的最大粘附量高于溶藻弧菌;9株拮抗菌的分离常数都高于溶藻弧菌;R32的亲和指数大于溶藻弧菌,其余8株拮抗菌的亲和指数都小于溶藻弧菌。测定了9株拮抗菌对病原性溶藻弧菌粘附大黄鱼表皮粘液的竞争、排斥和置换作用,结果显示:9株拮抗菌在竞争条件下都能极显著减少溶藻弧菌对表皮粘液粘附(P<0.01);在粘附排斥方面,R4、R13、J26、J28、J312、Y59等6株拮抗菌能显著排斥病原性溶藻弧菌对大黄鱼表皮粘液的粘附作用(P<0.05);在粘附置换方面,J26、J28等2株拮抗菌能极显著(P<0.01)降低病原性溶藻弧菌对大黄鱼表皮粘液的粘附量。研究结果表明,病原性溶藻弧菌对大黄鱼表皮粘液有较强的粘附作用;9株拮抗菌能够抑制溶藻弧菌对大黄鱼表皮粘液的粘附作用,其中竞争作用效果最好,排斥作用次之,置换作用的效果最差;拮抗菌对病原菌粘附的抑制效果受多种因素的影响,各菌株的粘附动力学参数有重要的影响作用。     

病原分离鉴定及致病性研究

于2013年4月从宁德患内脏白点病大黄鱼(Pseudosciaena crocea)中分离得到两株优势菌NZBD9和NZBD11, 这两株菌在16—19°C条件下回归感染能引起大黄鱼内脏白点病, 而在7—10°C和24—27°C条件下同样的人工感染不能致病, 从而证实这两株菌为大黄鱼内脏白点病的病原菌。经16S rDNA基因的测序和时间飞行质谱微生物鉴定仪分析, NZBD9 和NZBD11同为变形假单胞菌。药敏性实验结果显示NZBD9对庆大霉素、诺氟沙星和四环素等7种药物高度敏感。组织病理学观察结果显示病鱼的肝脏、脾脏、头肾等组织中均出现明显病症, 如变性和坏死。     

Vaccination in three different ways against vibriosis of Seriola dumerili caused by Vibrio hollisae

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili (amberjack) suffering from vibriosis. Healthy S. dumerili were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1:40 at 7 d, and reached its peak of 1:320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of <1:10, 1:20, 1:160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities (P<0.05). Upon challenge with virulent strain, the relative percent survival (RPS) of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.     

复方中药及其与抗生素联用对斜带石斑鱼变形假单胞菌病的防治效果

通过药物体外抑菌效果及其对斜带石斑鱼(Epinephelus coioides)抗病力影响的研究,筛选出有效治疗斜带石斑鱼变形假单胞菌(Pseudomonas plecoglossicida)病的复方中药与中西药联用配方。在不同复方中药实验组投喂饲料中添加不同比例黄连、黄芪与黄柏,设定三联复方中药实验组Ⅰ、Ⅱ、Ⅲ（黄连、黄芪、黄柏质量比分别为0.9∶〖KG-*2〗1.3∶〖KG-*2〗0.9、1.2∶〖KG-*2〗0.9∶〖KG-*2〗1.0、1.0∶〖KG-*2〗1.0∶〖KG-*2〗1.0）;复方中药与抗生素联用组中, 将不同质量比的中药黄连、黄芪、黄柏与抗生素恩诺沙星添加入饲料,分别设定为中西药联用实验组Ⅳ、V、VI。各三联中药与中西药联用实验组均添加占饲料2.2%质量分数的药物。研究结果为：三联复方中药浓度为60 mg/mL,黄连、黄芪、黄柏质量比为1.2∶〖KG-*2〗0.9∶〖KG-*2〗1.0时,抑制变形假单胞菌的效果最好,为复方中药组中最佳配方。养殖实验进行至40 d时,鱼死亡率由高到低分别为：对照组Ⅱ>对照组Ⅳ>对照组Ⅲ>实验组Ⅰ>实验组Ⅲ>实验组Ⅱ>实验组V>实验组VI >实验组Ⅳ>对照组Ⅰ。实验组Ⅱ（饲料中添加质量比为1.2∶〖KG-*2〗0.9∶〖KG-*2〗1.0的黄连∶〖KG-*2〗黄芪∶〖KG-*2〗黄柏）为药效最好的复方中药配方,实验组Ⅳ（黄连∶〖KG-*2〗黄芪∶〖KG-*2〗黄柏∶〖KG-*2〗恩诺沙星比为 0.9∶〖KG-*2〗1.3∶〖KG-*2〗0.9∶〖KG-*2〗1.3）为治疗鱼病效果最好的中西药联用配方。中西药联用相比复方中药治疗斜带石斑鱼变形假单胞菌病效果好,对于患变形假单胞菌病的斜带石斑鱼有较好的疗效。     

海洋微生物对甲胺磷农药的降解作用

作者从沿岸海域分离了38株有机磷农药的耐药菌,用分批培养法进行富集培养,得到有机磷农药的降解菌,并着重研究了其中两株菌对甲胺磷农药的降解情况,其结果表明,在10d内,降解菌株1对甲胺磷的降解率为59．1％,降解菌株2的降解率为53．3％,在各自的安磷培养液中,降解菌株1的数量大于降解菌株2;在甲胺降解过程中,其毒性逐渐减弱,降解菌株1所引起的毒性下降幅度大于降解菌株2。     

大黄鱼（Pseudosciaena crocea）一种新型抗菌肽的纯化及其生物学特性研究

以致病性溶藻弧菌为指示菌,通过酸粗提、固相萃取、Sephadex G-25柱凝胶层析和RP—HPLC等分离纯化技术,从大黄鱼胃肠组织中分离纯化抗菌肽。结果表明,大黄鱼肠道组织中存在着多种抗菌物质,并最终得到一种高纯度的新型抗菌肽AMP-W3。AMP—W3由10种氨基酸组成,其中天冬氨酸、谷氨酸和甘氨酸的含量较高。AMP...     

A Fast and Indriect Fluorescent Antibody Assay for the Vibro in Large Yeelow Croaker Pseudosciaena Crocea (Richardson)

A fast and indirect fluorescnet antibody assay for the Vibrio alginolyticus and V.Parahaemolyticus infecting the large yellow croaker has been developed.The specific antisera for the two strains of vibrio were prepared with New Zealand rabbit and the antiserum and cross-reactive efficacy was tested by coagulation in tube.It showed that the goat anti-rabbit IgG had been labeled by fuorescence isothiocyanate(FITC).The results showed that positive reactions were 100% for the large yellow croaker Pseudosciaena crocea with typical symptom of vibrio infection,while the positive reaction to the pathogen in healthy yellow croakers reached 40%,but seemed negative for aquaculter water.The results demonstrated that this fast and indirect fluorescent antibody assay can be used not only to test the vibrio pathogen in diseased yellow croaker but also in infected animals with no symptom.     

A Fast and Indirect Fluorescent Antibody Assay for the Vibrio in Large Yellow Croaker Pseudosciaena crocea (Richardson)

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Non-specific immune response of bullfrog Rana catesbeiana to intraperitoneal injection of bacterium Aeromonas hydrophila

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups.Each bullfrog in bacterium-injected group was injected intraperitoneally(i.p.) with 0.2 ml bacterial suspension at a density of 5.2 × 106 CFU/ml,while each one in control group injected i.p.with 0.2 ml sterile saline solution(0.85%,w/v).Three bullfrogs in both groups were sampled at 0,1,3,7,11,15 and 20 days post-injection(dpi) for the evaluation of non-specific immune parameters.It was observed that intraperitoneal injection of A.hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood.Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group.However,a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes.No significant difference in changes in the number of peripheral erythrocytes,serum superoxide dismutase(SOD) activity,and lysozyme activity was detected between the two groups.It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A.hydrophila infection.     

中草药复方及其与抗生素联用对斜带石斑鱼河流弧菌病的治疗效果

【目的】通过药物体外抑菌试验及斜带石斑鱼抗病力研究,筛选有效治疗斜带石斑鱼(Epinephelus coioides)河流弧菌(Vibrio fluvialis)病复方中药与中西药联用配方。【方法】用黄连、乌梅、黄柏比例分别为0.9∶1.3∶0.9、1.2∶0.9∶1.0、1∶1∶1的复方中药对河流弧菌进行体外抑菌实验;将质量分数2.2%复方中草药与中西药联用复方分别添加到基础饲料中,投喂感染河流弧菌的斜带石斑鱼73 d,研究中西药对石斑鱼河流弧菌病治疗效果。其中,试验组Ⅰ、Ⅱ、Ⅲ的黄连、乌梅、黄柏比例分别为0.9∶1.3∶0.9、1.2∶0.9∶1.0、1∶1∶1,试验组Ⅳ、Ⅴ、Ⅵ的黄连、乌梅、黄柏、恩诺沙星配比分别为0.9∶1.3∶0.9∶1.3、1.2∶0.9∶1.0∶0.9、1∶1∶1∶1,另设空白对照组Ⅰ(健康鱼,投喂基础饲料)、Ⅱ(病鱼,投喂基础饲料)和阳性对照组Ⅲ(病鱼,投喂板黄散)、Ⅳ(病鱼,投喂大黄五倍子散)。【结果】黄连、乌梅、黄柏配比1∶1∶1、质量浓度200mg/mL的复方中药对河流弧菌的体外抑菌效果较佳,为最佳配方及用量。40 d病鱼死亡数,对照组Ⅱ对照组Ⅳ对照组Ⅲ试验组Ⅰ试验组Ⅲ试验组Ⅱ试验组Ⅴ试验组Ⅵ试验组Ⅳ对照组Ⅰ,试验组Ⅱ的黄连、乌梅、黄柏比1.2∶0.9∶1.0为疗效最佳的中药复方,试验组Ⅳ的黄连、乌梅、黄柏、恩诺沙星比为0.9∶1.3∶0.9∶1.3为疗效最佳的中西药联用复方。【结论】中药复方及中西药联用复方均可较好治疗斜带石斑鱼河流弧菌病,后者药效优于前者。