草鱼肝微粒体的提取及CYP酶活性的测定

CYP酶代谢是药物生物转化的主要途径,其数量和活性大小直接影响药物在体内的活化与代谢。我们对草鱼肝微粒体CYP酶含量及其活性进行了初步研究,以差速离心法提取草鱼肝微粒体,以CO还原差示光谱法测得CYP酶及细胞色素b5含量分别为0.619±0.102 nmol/mg、0.264±0.042 nmol/mg。以7-乙氧异吩噁唑酮-O-脱乙基反应、苯胺-4-羟化反应、氨基比林-N-脱甲基反应作为CYP1A、CYP2E、CYP3A的探针反应,测得EROD酶活为0.043±0.004 nmol/mg/min,ANH酶活为0.028±0.002 nmol/mg/min,AMND酶活为0.207±0.035 nmol/mg/m in。结果表明草鱼肝微粒体中CYP酶发育完好,并且具有参与药物代谢的3种主要亚型活性,其含量与活性大小与其它实验动物相差较大。本实验的方法与结果为草鱼CYP酶的系统研究提供可靠手段,最终为指导水产合理用药提供理论依据。     

Hepatic P450 monooxygenase response in rainbow trout (Oncorhynchus mykiss (Walbaum)) administered aquaculture antibiotics

The effects of the 4-quinolones, oxolinic acid and flumequine on hepatic microsomal cytochrome P450 monooxygenases in rainbow trout were examined during this study. Following antibiotic administration in the diet for 10 days at a representative commercial medicated feed concentration, fish were killed for assay at various periods up to 12 days following a return to normal diet. Ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were significantly elevated as a result of antibiotic treatment. The effects of oxolinic acid were delayed and longer-lasting compared with flumequine. The effects of flumequine were detectable 4 days earlier than those of oxolinic acid and lasted for less than ten days after treatment cessation. In contrast, oxolinic acid effects were apparent even on day 12 of the recovery period. The results of O-dealkylation, isoform-selective inhibition, and immunoblotting showed that the effects of both oxolinic acid and flumequine were related to P4501A subfamily. P450-binding spectra and experiments in vitro suggested that both antibiotics are weak dose-independent inhibitors of P450 activity, with flumequine exhibiting slightly higher binding affinity and inhibitory activity.     

The cytochrome P450 system of Atlantic salmon (Salmo salar): II. Variations in hepatic catalytic activities and isozyme patterns during an annual reproductive cycle

A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5–7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17 was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b₅, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b₅, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17 levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17 as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17 independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goks\/oyr 1989).     

Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain

Cytochrome P450 (CYP) 1A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP 1A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP 1A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by β-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP 1A1 antibodies were used. ELISA measurements confirmed the induction of CYP 1A1 protein in the rainbow trout brain subcellular fractions.     

福寿螺细胞色素P450基因CYP3192A1的克隆与表达分析

文章分析了福寿螺(Pomacea canaliculata)细胞色素P450(CYPs)的结构、表达特征及四聚乙醛代谢与细胞色素P450表达水平的相关性。应用c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)克隆获得福寿螺CYPs基因c DNA全长2 523 bp,命名为CYP3192A1,含1个开放阅读框(ORF),编码517个氨基酸,具有血红素结合区F**G***C*G、K螺旋(E**R)、I螺旋(AG*ET)、C螺旋(W***R)等高度保守序列及跨膜螺旋(12~34号氨基酸)。聚类分析显示福寿螺CYP3192A1是细胞色素P450新家族成员,与CYP3A亚家族序列同源性较高。荧光定量PCR(real time quantitative PCR,RT-q PCR)分析显示,福寿螺CYP3192A1基因表达量存在显著的性别及组织差异性(P0.05),表现为雌性表达量高于雄性,肠和鳃表达量最丰富,肝和胃次之,心和腹足中少量表达;四聚乙醛对福寿螺CYP3192A1表达的影响为"低浓度诱导,高浓度诱导期缩短并提前"。结果表明四聚乙醛处理能够显著诱导福寿螺CYP3192A1表达,与CYP3192A1表达量增加紧密相关,福寿螺CYP3192A1过表达可能在四聚乙醛代谢抗性中起重要作用。     

Plasmid‐mediated antimicrobial resistance in motile aeromonads from diseased Nile tilapia (Oreochromis niloticus)

For the sustainable farming of tilapia, proper maintenance of their health and adequate treatment for infections at appropriate time are inevitable. The indiscriminate use of antibiotics in aquaculture, as a part of treatment and as growth promoters, accelerates antimicrobial resistance (AMR) among the fish pathogens. In the present study, we have isolated diverse aeromonads from Nile tilapia and studied their antibiogram and plasmid profiling. Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Aeromonas dhakensis, Aeromonas caviae, Aeromonas jandaei and Aeromonas aquatica were isolated from infected tilapia (n = 150), and their Shannon wiener diversity index was calculated as 1.926. A. veronii was found to be the most multiple antibiotic‐resistant pathogen with the MAR index of 0.46, and A. aquatica was noticed as the least resistant isolate. The minimum inhibitory concentration of resistant antibiotics was shown as >256 mcg/ml for most of the isolates. The virulent genes such as aerolysin and hemolysin were identified in all the isolates except A. aquatica. The detection of class 1 integrons, plasmid profiling and plasmid curing studies confirmed that AMR exhibited by most of the Aeromonas species is of plasmid mediated. This challenges the risk of wide spread of AMR among the pathogens and subsequent treatment of the infection.     

High prevalence of antimicrobial resistance in Escherichia coli,Salmonella spp. and Staphylococcus aureus isolated from fish samples in India

Antibiotic resistance, driven by inappropriate use of antimicrobial agents for fish farming, is an expanding global health threat. Demand of animal protein for human consumption is entraining at remarkable pace which in turn, upturning the non‐judicial use of life‐saving drugs in these modern animal production practices at an incautious rate. Hence, study was devised to observe the antimicrobial resistance (AMR) pattern of human prominence bacteria, against drugs of critically and highly importance of both human and veterinary medicine, circulating over the fish farming in India. One hundred sixty fish faecal samples were obtained from distinct geographical locales of Rajasthan, India. Overall, 202 isolates comprising of Escherichia coli (81, 51%), Salmonella spp. (72, 45%) and Staphylococcus aureus (49, 31%) were isolated to screen for resistance. Antimicrobial resistance has been analysed using broth microdilution method. In case of E. coli and Salmonella spp., highest resistance (>95%) was observed against streptomycin while for S. aureus, it was observed against trimethoprim, along with high resistance to other antibiotics. Majority of the isolates were found to be resistant to more than three antibiotics implying multi‐drug resistance (>89%) relative to the critical and highly important antibiotics. The high AMR is a reflection of misuse of these agents in the fish farming, which is a concern of serious health issues. Our findings persuade the prudent use of antimicrobial agents in fish farming and other aquaculture settings that may assist in drafting of the new policies for the judicial use of these life‐saving drugs in these sectors.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

剑尾鱼脑细胞系的建立及细胞色素P4501A的诱导表达

为了构建剑尾鱼脑细胞系并探讨其细胞色素P4501 a(CYP1A)基因的诱导效应,实验通过胰蛋白酶消化法对剑尾鱼脑组织进行体外培养,经连续继代培养,建立了可稳定传代的脑细胞系,命名为SFB.SFB最适培养液为含有15％胎牛血清(FBS)的DMEM/F-12和L-15等比混合培养液,培养条件为27℃,5％ CO2.生长特性研究表明,第65代细胞的群体倍增时间为43.048h,显示出旺盛的生长和分裂能力.染色体分析发现,培养细胞的染色体众数为48条,SFB核型公式为2n =2st +46t,臂指数(NF) =48,与剑尾鱼一致.诱导实验表明,SFB在10-8～ 10-5 mol/L的苯并(a)芘诱导下,CYP1A mRNA表达量显著提升,且表现出良好的剂量效应关系.脑细胞系的建立为剑尾鱼的毒理学评价研究提供了便利,也为其系统的生态毒理学应用打下基础.     

Synergistic effect of sodium selenite and Pediococcus acidilactici on growth,intestinal bacterial counts,selenium bioavailability,hepatic enzymes and non‐specific immune response in rainbow trout (Oncorhynchus mykiss)

Lactic acid bacteria (LAB) have a crucial role in inorganic selenium metabolism as well as their known desirable effects on fish. In this study, the synergistic effects of dietary sodium selenite and Pediococcus acidilactici on growth performance, intestinal bacterial counts, selenium bioavailability, hepatic antioxidant enzyme thioredoxin reductase activity and hepatic glycolytic enzyme activity that is hexokinase, phosphofructokinase and pyruvate kinase, and non‐specific immune response such as serum lysozyme and complements C₃, C₄ and ACH₅₀ activity in rainbow trout (Oncorhynchus mykiss) were investigated. Thus, a 3 × 3 factorial design experiment was conducted with nine purified diets including three levels of sodium selenite (0, 1 and 2 mg/kg) and three levels of P. acidilactici (0, 7 and 9 log CFU/g). After 8 weeks of feeding, weight gain and specific growth rate were increased by increasing dietary sodium selenite and P. acidilactici levels compared to control (p < .05), whereas feed conversion ratio and condition factor was decreased by increasing dietary sodium selenite and P. acidilactici amounts in comparison with control (p < .05). Survival rate was not significantly affected among the experimental treatments (p > .05). Total cultivable bacterial populations after 4 and 8 weeks of the feeding trial were not significantly different among the dietary treatments, while LAB levels were higher in P. acidilactici‐fed groups than in control and selenium‐fed groups (p < .05). Selenocysteine, methylselenocysteine and selenomethionine levels in the intestine of rainbow trout were increased by increasing the sodium selenite and P. acidilactici levels (p < .05), and selenocysteine was found the most selenium species in the trout intestine. The quantity of total selenium in the whole body, intestine, blood, liver and muscle of rainbow trout were increased by increasing the amounts of sodium selenite and P. acidilactici compared to control (p < .05). Hepatic thioredoxin reductase and hexokinase activity were increased by increasing dietary selenium and P. acidilactici levels in comparison to control (p < .05), whereas phosphofructokinase and pyruvate kinase activity in the liver of rainbow trout were not significantly different between the dietary treatments. Serum lysozyme, complements C₃, C₄ and ACH₅₀ activity were enhanced by increasing dietary selenium and P. acidilactici levels compared to control (p < .05). The most synergistic effects of dietary supplements on growth and metabolism of rainbow trout were obtained at 2 mg/kg sodium selenite and 7 log CFU/g P. acidilactici. The findings revealed the synergistic effect of dietary selenium and P. acidilactici on growth and metabolism in rainbow trout (O. mykiss).     

Acute responses of juvenile cobia Rachycentron canadum (Linnaeus 1766) to acid stress

Fish are potentially submitted to water acidification when reared in recirculating aquaculture systems. This study evaluated the responses of juvenile cobia Rachycentron canadum after acute exposure to acid water. Juvenile cobia (12.6 ± 0.5 g; 14.2 ± 0.2 cm) were acutely exposed to four pH levels (7.9 (control), 6.5, 6.0, and 5.5). After 24 h of exposure to different pH values, fish were sampled for physiological and histopathological evaluation. Acid water affected physiological parameters and induced morphological histopathologies on gill and skin of juvenile cobia, and these effects were more conspicuous with decreasing pH values. Acid stress induced blood acidosis in juvenile cobia, coupled to a decrease in bicarbonate (HCO₃^?) and saturated O₂ (sO₂) in fish blood. On the other hand, haematocrit, haemoglobin and glucose concentration increased their values (P < 0.01) comparing to control level. Hyperplasia with completely fusion of secondary lamella was observed in all pH treatments (6.5. 6.0 and 5.5), while telangiectasia and proliferation of chloride cells were present for fish exposed to pH 6.0 and 5.5. In skin hyperplasia and hypertrophy of mucous cells, necrosis of these cells for fish exposed to pH 6.0 and 5.5 was observed. The results of this study demonstrate that acute acid water exposition affected physiology and histopathology in juvenile cobia, especially at pH values below 6.5. Accordingly, particular attention must be given to pH during cobia reared in recirculating aquaculture.     

草鱼孕烷X受体与细胞色素P450 3A mRNA表达相关性初步研究

根据GenBank中斑马鱼(Danio rerio)和虹鳟(Oncorhynchus mykiss)孕烷X受体(pregnane X receptor,PXR)基因序列设计保守区域简并引物,通过PCR扩增获得草鱼(Ctenopharyngodon idellus)PXR cDNA核苷酸序列片段为509 bp,经推导获取169 aa序列。对草鱼与其它物种PXR氨基酸序列进行同源性比较,用以鉴定其在物种中的进化。应用实时荧光定量PCR(qRT-PCR)检测草鱼9种组织PXR和细胞色素P450 3A(cytochrome P450 3A,CYP3A)基因mRNA相对含量,结果表明,其表达丰度依次为:前肠﹥肝脏﹥肾脏﹥鳃﹥肌肉﹥后肠﹥性腺﹥心脏﹥脾脏,CYP3A和PXR基因在草鱼组织中表达分布基本一致,在前肠、肝脏和肾脏高度表达,而在其它组织低度表达。为了进一步研究PXR和CYP3A基因表达相关性,通过细胞,特选用诱导剂利福平(rifampicin,RIF),最佳诱导浓度40μM,孵育1、2、4、6、8、10、12、24 h后,用qRT-PCR检测CYP3A-PXR基因动态表达过程。结果显示,诱导组CYP3A和PXR基因表达量均高于对照组,显示出显著诱导效应。     

Stress response of Salmo salar (Linnaeus 1758) facing low abundance infestation of Caligus rogercresseyi (Boxshall & Bravo 2000), an object in the tank,and handling

This study looks at how low infestation loads of adult Caligus rogercresseyi and other stressors affect the physiology of Salmo salar. Experimental fish groups were with (infested) or without (control) exposure to the parasite. The parasite cohort was followed for 78 days post‐infestation (dpi), and only adult lice were observed. Additional stressors were applied at 60 and 75 dpi. The analysis included measurements of fish physiology and weight. Low‐level infestations by adult C. rogercresseyi for more than 50 dpi induced moderate stress in S. salar as well as a high energy demand and increased small skin mucous cells. Threshold lice loads were identified, and above those loads, a high stress response was observed. Additional stressors altered fish physiology, inducing downregulation of the cortisol response after the first stressor and upregulation after the second stressor, but infested fish responded more strongly. Parasitism by C. rogercresseyi is energetically demanding, affecting the primary and secondary responses (e.g. cortisol and glucose levels), as well as the tertiary response (fish weight).     

Growth parameters,innate immune response and resistance to Listonella (Vibrio) anguillarum of Dicentrarchus labrax fed carvacrol supplemented diets

The research was aimed to assess the effect of dietary carvacrol (0.025% and 0.05%) on sea bass (Dicentrarchus labrax) growth, immune response and resistance to Listonella anguillarum. Fish (69.2 ± 0.22 g) were fed the experimental diets for 9 weeks. Dietary carvacrol did not negatively affect fish survival, growth performance, feed intake and feed conversion ratio (P > 0.05) nor carcass yield and viscerosomatic, hepatosomatic and mesenteric fat index (P > 0.05). Serum and head kidney leucocytes were collected after 1, 4 and 8 weeks of feeding. Carvacrol significantly reduced serum proteins, immunoglobulins and lysozyme activity (P < 0.01) and moderately increased phagocytosis and pinocytosis of head kidney macrophages. The release of reactive oxygen species by leucocytes was reduced in carvacrol‐fed fish, even if significantly (P < 0.05) only in those fed 0.05% carvacrol for 1 week. Dietary carvacrol did not significantly affect the aspecific immune response, although a potential antioxidant activity might be speculated. Moreover, feeding carvacrol provided an appreciable resistance to a challenge with L. anguillarum, when a bacterial dose lower than the Lethal Dose₅₀ was used. Cumulative mortality in fish fed 0.025% carvacrol was significantly lower than that of untreated controls (75% Relative Per cent Survival).     

Influences of dietary antimicrobial peptide APSH‐07 on the growth performance,immune response and vibriosis resistance of abalone Haliotis discus hannai Ino

A 115‐day feeding trial and subsequently a 10‐day challenge test with Vibrio parahaemolyticus were conducted to investigate the effects of dietary antimicrobial peptide APSH‐07 on growth performance, immune response, antioxidative status and vibriosis resistance of abalone Haliotis discus hannai (initial body weight: 2.06 ± 0.01 g; initial shell length: 25.42 ± 0.18 mm). Four artificial diets were designed with 0 (artificial diet control), 7.5, 15.0 and 22.5 mg/kg of APSH‐07, respectively. The brown alga Laminaria japonica was used as the live food control. Results showed that the specific growth rates of abalone in the groups with 7.5 and 15.0 mg/kg dietary APSH‐07 were significantly higher than those in the controls (p < .05). The total haemocyte counts and respiratory burst activity in haemolymph of abalone in the group with 7.5 mg/kg of dietary APSH‐07 were significantly higher than those in the groups with 0 and 22.5 mg/kg of dietary APSH‐07 (p < .05). The gene expression levels of Mn‐superoxide dismutase and thioredoxin peroxidase 2, and the activities of glutathione peroxidase in the group with 7.5 mg/kg of dietary APSH‐07 were significantly higher than those in the other groups (p < .05). Cumulative mortality of abalone after the challenge test was significantly decreased in the group with 7.5 mg/kg of dietary APSH‐07 supplementation. Supplementation of 22.5 mg/kg dietary APSH‐07 significantly increased the cumulative mortality. In conclusion, 7.5 mg/kg of dietary APSH‐07 supplementation had the better growth performance, higher antioxidation, immune and disease resistance capacity of abalone. Excessive supplementation of dietary antimicrobial peptide APSH‐07 (22.5 mg/kg) had significantly negative effects. Further studies are needed to determine the optimal level of dietary APSH‐07 supplementation for abalone.     

Exophiala angulospora infection in hatchery‐reared lumpfish (Cyclopterus lumpus) broodstock

Samples from moribund lumpfish were collected in a marine hatchery in Scotland in 2015. Black nodules were noted on the skin, and gills and fungal hyphae were extensively distributed in musculature and internal organs. Multifocal chronic inflammatory lesions displaced structures in all affected organs. Mortalities commenced on completion of spawning in May and were evenly distributed over the second year in the temperature range 11–15°C. The main systemic infection causing agent was initially identified based on morphological characteristics as an Exophiala species. Ribosomal DNA (rDNA) ITS regions of the isolates were subsequently sequenced confirming the isolates belonged to Exophiala genus. All isolates fell in a single phylogenetic cluster, which is represented by Exophiala angulospora. Fish were treated with either formalin or Bronopol or a combination of both, but there was no effect on the pattern or numbers of mortalities. Isolates were also tested against three different concentrations of Latrunculin A, Amphotericin B and Itraconazole with no success. It is of utmost importance to increase the knowledge on pathogen–host interactions to successfully develop sustainable control methods.