In vitro comparisons of the inhibitory activity of florfenicol,copper sulphate and potassium permanganate towards Aeromonas hydrophila and Flavobacterium columnare

Aeromonas hydrophila and Flavobacterium columnare, the aetiological agents of motile aeromonas septicaemia (MAS) and columnaris disease respectively, have been recently causing crippling mortalities to the sunshine bass, Morone chrysops female ×Morone saxatilis male (Percichthyidae), industry in the United States. Isolates of A. hydrophila and F. columnare obtained from fish that died during farm outbreaks were subjected to in vitro evaluation of florfenicol (FFC), copper sulphate (CuSO₄) and potassium permanganate (KMnO₄). Florfenicol inhibited the growth of A. hydrophila and F. columnare more than CuSO₄ and KMnO₄. The minimum inhibition concentration (MIC) of FFC was 0.04 ± 0 and 0.2 ± 0.1 mg L^?1 for A. hydrophila and F. columnare respectively, while the 50% inhibition concentration (IC50) for A. hydrophila and F. columnare was 0.23 ± 0.01 and 0.4 ± 0.2 mg L^?1 respectively. Copper sulphate was more effective against A. hydrophila than KMnO₄; CuSO4 had a MIC of 83.2 ± 0 mg L^?1 compared to 158.0 ± 0 mg L^?1 for KMnO₄. Copper sulphate was also more effective against F. columnare than KMnO₄. The IC50 values of CuSO₄ and KMnO₄ towards F. columnare were 4.8 ± 0.3 and 8.7 ± 1.6 mg L^?1 respectively, and the minimum bactericidal concentration values of CuSO₄ and KMnO₄ towards F. columnare were 25.0 ± 0 and > 158.0 mg L^?1 respectively. In addition, F. columnare was more sensitive to CuSO₄ and KMnO₄ than A. hydrophila.     

In vitro and in vivo antibacterial activity of shrimp chitosan against some intestinal bacteria

ABSTRACT:   The effects of shrimp chitosan with deacetylation degrees (DD) of 50%, 70% and 95% (DD50, DD70, DD95) on the growth of the intestinal bacteria were investigated in vitro in the laboratory media, and in vivo by an oral feeding test using hamsters as the animal model. The antibacterial activities of these chitosan products against one strain of pathogenic Clostridium perfringens and 13 strains of probiotics, including seven strains of Lactobacillus , and six strains of Bifidobacterium were evaluated. In vitro , the antibacterial activities of DD95 and DD70 were much higher than that of DD50. The strains of probiotics were more resistant to chitosan than the pathogen of C .  perfringens . The minimal lethal concentration for DD95 against C. perfringens was 250 p.p.m., whereas the survival percentages for most probiotics tested were above 90% for DD95 at 500 p.p.m. The animals were fed on either a control diet, or diets containing powdered chitosan instead of 5% cellulose in the control diet for 4 weeks. The cecal bacterial counts of total aerobes, total anaerobes, lactobacilli, bifidobacteria and clostridia were similar for the control and experimental groups. The reasons for the differences in the antibacterial activity in vitro and in vivo are discussed.     

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment‐friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon^?S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10‐fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.     

In vitro antibacterial activity of the combination of sulphadiazine and trimethoprim on bacterial fish pathogens

Abstract. The in vitro bacteriostatic activity of a combination of sulphadiazine (SDZ) and trimethoprim (TMP) against representative bacterial fish pathogens was studied. In general, the minimum inhibitory concentrations (MIC) of TMP were much smaller than comparable values for SDZ. When SDZ and TMP were combined, the bacteriostatic effect was enhanced in a synergistic manner for all bacteria tested except Pseudomonas fluorescens . The synergism was most remarkable in the case of bacterial strains that were sensitive to the action of SDZ alone. This degree of synergism was observed when the ratios of SDZ and TMP in combination were in the range of 1:1 io 5:1. The MIC of SDZ plus TMP combined was less than 3.13 μg/ml for SDZ resistant strains and as low as 0.2 to 0.78 μ/ml for SDZ sensitive strains. The possible usefulness of a combination of these two compounds for therapy or control of bacterial fish disease is discussed.     

Effects of photoperiod alterations on day-night variations in hypothalamic serotonin content and turnover,and monoamine oxidase activity in the female catfish,Heteropneustes fossilis (Bloch)

The effects of different photoperiod regimes, and total darkness on day-night variations in hypothalamic serotonin (5-HT) content and turnover index (TI), and monoamine oxidase (MAO) activity that occur exclusively during the gonadal preparatory phase (February–March) were investigated in female Heteropneustes fossilis. Exposure of the fish to long photoperiods (16L:8D; light between 06.00–22.00h, for 40 days elevated both the midphotophase and midscotophase values of 5-HT content and MAO, and abolished their day-night differences. The daily variations of 5-HT-TI was further intensified by the treatment. Under short photoperiods (4L:20D; light between 18.00–22.00h for 40 days), there was a significant decrease in both 5-HT content and TI, a reversal of the day-night variations of 5-HT content and MAO activity, and loss of the 5-HT-TI pattern. In fish maintained in total darkness the day-night variations were not found and there was a significant inhibition of 5-HT. Exposure of the fish to continuous light abolished the day-night variations of these correlates and elevated their values at both intervals. When the LD cycle was reversed, there was a corresponding shift in the day-night patterns. The gonadosomatic index (GSI) was significantly elevated in the long photoperiod and continuous illumination groups, significantly decreased in the short photoperiod and total darkness groups, and unchanged in the reversed LD cycle regime. The results indicate that the day-night variations of 5-HT and MAO are photoperiod-dependent and are controlled by the prevailing LD cycle during the gonadal preparatory phase of the annual reproductive cycle. The photoperiod effects on the gonadal activity may be mediated through the hypothalamic serotonergic system.     

6种中草药对4种淡水鱼致病菌体外抑菌作用的研究

研究了6种中草药对4种淡水鱼致病菌温和气单胞菌（Aerom onas sobria）、嗜水气单胞菌（A.hydrophila）、生物Ⅰ-荧光假单胞菌（Pseudom onas fluorescentb iotypeⅠ）和柱状黄杆菌（Flavobacterium columnaris）的体外抑菌作用。以黄连（Coptis chinensis）、大黄（Rhem officinale）、连翘（Forsythia suspensa）、鱼腥草（Houttuynia corda-ta）、厚朴（Magnolia officinalis）和大青叶（Folium isatidis）6种中草药的水提物为研究对象,采用滤纸片法检测其对4种致病菌的抑制活性,并分别测定了这6种植物提取物对供试菌的最小抑菌浓度（M IC）。结果表明,黄连水提物对4种淡水鱼致病菌的抑菌效果最好,大黄和连翘水提物次之,而鱼腥草、厚朴和大青叶水提物的抑菌效果不明显。     

Qualitative and quantitative identification of L-gulonolactone oxidase activity in some teleosts

Abstract. L-gulonolactone oxidase activity was assessed in kidney and liver tissues of 14 teleosts by use of a histochemical technique for qualitative identification of the presence of enzyme activity and a biochemical technique for quantification of activity of the enzyme. Nine tilapias, two cyprinids and three salmonids were investigated; enzyme activity was only detected in liver and kidney tissues of common carp, Cyprinus carpio L., and kidney tissue of Oreochromis spilurus (Günther) and O. aureus (Steindachner).     

血管紧张素转化酶抑制肽研究进展

为研究鳗鱼加工副产物的综合利用,以鳗鱼骨胶原蛋白为原料,采用酶解法制备血管紧张素转化酶 (Angiotensin-converting enzyme, ACE) 抑制肽。以ACE抑制活性和水解度为评价指标,通过单因素和响应面试验确定最佳的酶解制备条件,并分析酶解产物的分子质量分布和氨基酸组成。结果显示,碱性蛋白酶为最适水解酶,最佳酶解条件为：温度50 ℃,质量浓度15 g·L^–1,酶解时间5.25 h,加酶量3.1% (质量分数),pH 9.2。在此条件下,鳗鱼胶原蛋白肽的ACE抑制活性为70.33%,与预测值接近;酶解产物中分子质量小于1 kD的肽占57.02%,1~3 kD的肽占36.55%;氨基酸组成分析表明,酶解产物中与ACE抑制活性有关的疏水性氨基酸 (如脯氨酸、缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸) 含量增加。     

In vitro antibacterial activity of four nano‐encapsulated herbal essential oils against three bacterial fish pathogens

Bacterial pathogens causing diseases in fishes have broad host ranges, and may cause high mortality or ongoing chronic infections. Pathogen treatments using antibiotics are becoming limited due to increasing concerns for the development of resistance and environmental dissemination of bacteria harboring resistance genes. This study aimed to determine whether nano‐emulsions of selected plant‐derived essential oils (Origanum vulgare, Eucalyptus globulus, Melaleuca alternifolia and Lavendula angustifolia) were bacteriostatic or bactericidal to Aeromonas hydrophila, Streptococcus iniae and Photobacteriumdamselae subspecies damselae. All treatments showed antibacterial activity, and in almost all cases the activity of the nano‐emulsions was superior to their essential oil counterparts. Origanum vulgare (oregano) nano‐emulsion had the most effective antibacterial activity, with a minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 3.12 µg/ml against all three species; substantially better than tetracycline. The present study highlights the potential of new formulations of essential oils to enhance bacterial disease control in cultured fish.     

Xanthine oxidase activity exerts pro‐oxidative and pro‐inflammatory effects in serum of silver catfish fed with a diet contaminated with aflatoxin B1

Several studies have associated the involvement of xanthine oxidase (XO) activity, a source of uric acid and reactive oxygen species (ROS), to pro‐oxidative and pro‐inflammatory effects during pathological conditions. Considering this, the aim of this study was to evaluate whether upregulation on seric XO activity may be a pathway involved in the oxidative stress in fish exposed to a diet contaminated with aflatoxin B₁ (AFB₁), as well as whether it may be considered a pathway involved in ROS and NOx production. Xanthine oxidase activity, as well as the uric acid, ROS and NOx levels increased in serum of fish fed with a AFB₁‐contaminated diet on days 14 and 21 post‐feeding compared to fish fed with a basal diet. Based on these evidences, upregulation of seric XO activity induces pro‐oxidant and pro‐inflammatory profiles in serum of fish fed with a AFB₁‐contaminated diet due to excessive formation on uric acid. Also, the excessive uric acid induces the release of pro‐oxidant and pro‐inflammatory mediators, as ROS and NOx, also contributing to oxidative and inflammatory profiles. In summary, the upregulation on seric XO activity may be considered a pathway involved in the oxidative stress of fish exposed to a diet contaminated with AFB₁.     

鳄鱼血蛋白酶解产物抗氧化特性和血管紧张素转化酶抑制活性研究

研究了鳄鱼血蛋白酶解产物的抗氧化特性和对血管紧张素转化酶（ ACE）的抑制活性。利用木瓜蛋白酶酶解鳄鱼血浆蛋白和血球蛋白,用分光光度法测定了酶解产物的抗氧化能力和用高效液相色谱（ HPLC）测定其ACE的抑制率。结果显示：鳄鱼血浆和血球蛋白酶解产物的亚铁离子螯合能力差异性不显著（ P＞0．05）;在0～5 mg/mL的浓度范围内,血球蛋白酶解产物清除ABTS自由基的能力大于血浆蛋白酶解产物,且在浓度为1 mg/mL时,两者清除ABTS自由基的能力差异性极显著（ P＜0．01）;血浆蛋白酶解产物清除DPPH自由基的能力在0～5 mg/mL的浓度范围内随着蛋白浓度的增加而升高,血球蛋白酶解产物在蛋白浓度为4 mg/mL处达到最大清除率,之后下降;在0～20 mg/mL的浓度范围内,两种酶解产物的还原力随着蛋白浓度的提高显著升高,但两者还原力的差异性不显著（ P＞0．05）;鳄鱼血浆和血球蛋白酶解产物对ACE具有良好的抑制力,其最大抑制率可分别达到75．56％和86．42％。研究表明,鳄鱼血蛋白酶解产物在体外具有抗氧化和抑制ACE的活性。     

海洋细菌Phaeobacter DL2的鉴定及其对致病弧菌抑制作用

从海水养殖水体中防腐钢片的微生物黏膜上分离筛选到一株细菌,命名为DL2.采用琼脂扩散法进行体外抑菌实验,测定其抑菌谱,发现菌株DL2具有较广泛的抑菌谱,并且对致病性弧菌表现出较强的抑菌作用.通过常规生理生化研究和细菌鉴定系统测试,发现菌株DL2属于革兰氏阴性菌,短杆状,具极生单鞭毛,菌落呈褐色,能产生浅褐色扩散性色素,测试结果表明,菌株DL2与Phaeobacter属细菌的特征非常相似.为了进一步确定菌株DL2的分类学地位,测定了其16S rRNA基因序列,与相关细菌种属相应序列的同源性进行比较并构建了系统关系树,结果显示,菌株DL2与Phaeobacter inhibens的亲缘关系最近,相似性达99.8%,与Phaeobacter gallaeciensis相似性达99.1%.综合上述结果,菌株DL2可鉴定为Phaeobacter inhibens,同时可以将其作为潜在的海洋有益菌应用于水产养殖中.     

Hepatic 5′-monodeiodinase activity in teleosts in vitro: A survey of thirty-three species

The in vitro hepatic 5′-monodeiodination of thyroxine (T₄) to triiodothyronine (T₃) in Oreochromis mossambicus, Channa striata, Clarias batrachus, Cyprinus carpio and Oxyeleotris marmorata was found to be time, pH and temperature dependent, and related to the amount of substrate (T₄) and homogenate introduced into the reaction vessel, in a manner which was consistent with Menton-Michaelis kinetics, and thus indicative of an enzyme-regulated process. Dithiothreitol introduced into the reaction vessel stimulated T₃ production in a dose-related manner. Hepatic 5′-monodeiodinase activity was also detected in a further 28 species of teleosts suggesting that the peripheral monodeiodination of T₄, which is well-documented in salmonids, is also widespread amongst other teleost fishes. All species examined exhibited evidence of enzymatic deiodination, but there were marked differences in K_m and V_max values between the species. There was no apparent phylogenetic or environmental relationships to explain the widely divergent K_m and/or V_max values, nor was there a correlation between K_m and V_max when the species were considered together.     

In vitro activities of oxolonic acid, ciprofloxacin and norfloxacin against Aeromonas salmonicida

Abstract. Oxolinic acid and two new fluoroquinolones, ciprofloxacin and norfloxacin, were evaluated for in vitro antibacterial activity against Aeromonas salmonicida. Although oxolinic acid was as active as ciprofloxacin in terms of minimum inhibitory concentration (MIC), fluoroquinolones were significantly more active in terms of their ability to kill both oxolinic acid sensitive and resistant strains of A. salmonicida. Furthermore, the fluorinated drugs were active against non-dividing A. salmonicida. It would appear worthwhile to carry out further investigations with fluoroquinolones as they may be more effective in treating A. salmonicida infections than the current regime of oxolinic acid.     

In vitro metabolism of progesterone, androgens and estrogens by rainbow trout embryos

The purpose of the study was to determine the steroid metabolic pathways used by rainbow trout (Oncorhynchus mykiss) embryos. Whole embryo preparations at 42, 53 and 65 days post fertilization (dpf) were incubated, in vitro, with the tritium-labelled steroids, progesterone (P₄), testosterone (T), androstenedione (A4), 17β-estradiol (E₂), and estrone (E₁), and the metabolites formed were separated and identified by reverse phase high performance liquid chromatography (HPLC). The degree of metabolism for each of the substrates was dependent on the age of the embryos, and was always higher in older embryos. P₄ yielded three metabolites, one of them identified by gas chromatography-mass spectroscopy as 3β,7α-dihydroxy-5α-pregnan-20-one. Several unknown metabolites of a similar profile on HPLC were produced for T and A₄, in addition to A₄ and T as metabolites of T and A ₄ metabolism, respectively. Likewise, E₂ and E₁ yielded each other and an unknown polar metabolite. These observations argue for the presence of 5α-reductase, 7α-hydroxylase and 17β-hydroxysteroid dehydrogenase activity in embryo tissues which, by their conversion of biologically active yolk steroids of maternal origin, may provide a protective environment for the embryo during early development.     

In vitro antibacterial effect of berberine hydrochloride and enrofloxacin to fish pathogenic bacteria

Berberine the main antibacterial substance in the rhizome of Coptis chinensis Franch, is used as an antimicrobial agent for a long time. This study was carried out to investigate the potential of berberine alone or in combination with enrofloxacin against six common fish pathogens for use in fish disease management in aquaculture. The minimum inhibitory concentrations (MICs) of berberine hydrochloride against Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio vulnificus, Edwardsiella ictaluri, Escherichia coli and Streptococcus agalactiae were determined and to be >500, >500, >500, 300, 400 and 100 μg mL^?1 respectively. The minimal bactericidal concentrations (MBCs) of berberine hydrochloride to E. coli, E. ictaluri and S. agalactiae were 300–500 μg mL^?1. In combination with enrofloxacin, the MICs and MBCs of berberine hydrochloride significantly decreased against E. coli and E. ictaluri but not against Streptococcus dysgalactiae. These results demonstrated that the berberine hydrochloride enhanced the bactericidal effect of enrofloxacin and vice versa. The synergistic bactericidal effect of berberine hydrochloride and enrofloxacin suggest its potential use in fish disease management in aquaculture. This is the first study on the inhibitory effect of berberine hydrochloride against fish pathogenic bacteria.     

双齿围沙蚕化学成分及其浸膏抗肿瘤活性的研究

采用硅胶柱色谱及薄层制备色谱等手段从双齿围沙蚕(Perinereis aibuhitensis)中分离纯化出2种化合物。对所获得的化合物通过理化常数测定、波谱数据分析同文献对照的方法进行了结构鉴定。同时,采用CCK-8方法对双齿围沙蚕浸膏及乙酸乙酯萃取部分分别进行抑制肿瘤细胞活性测试。结果表明,从双齿围沙蚕中分离得到2种化合物分别为:胆甾-4烯3α,6β醇(1)和(E)-17-(4,7-dimethyloct-5-en-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol(2),且化合物1是首次从沙蚕中分离得到。抑制肿瘤细胞活性测试表明,70%乙醇浸提获得的双齿围沙蚕浸膏及乙酸乙酯萃取部分对人胃癌细胞AGS、人肝癌细胞HepG2以及人卵巢癌细胞SKOV3均具有抑制细胞增殖作用,且具有剂量和时间依赖性,可作为潜在的抗肿瘤药物进行进一步研究。