嗜水气单胞菌气溶素单克隆抗体的制备及初步应用

以1％福尔马林灭活的嗜水气单胞菌纯化的气溶素免疫8周龄的BALB／C小鼠，取其脾细胞与SP2／0细胞融合，经间接ELISA筛选，获得1株稳定分泌气溶素单克隆抗体的杂交瘤细胞，命名为A2D3。腹水单抗ELISA效价为1：16，384（1：2^14），其能特异性抑制气溶素的溶血活性并且腹水单抗有中和特性，抑制效价和中和效价分别为1：128（1：2^7）和1：128（1：2^7）。以此单抗建立了检测嗜水气单胞菌气溶素的问接ELISA方法，对临床分离的50株嗜水气单胞菌培养上清检测，有48株呈阳性反应。     

鱼源气单胞菌的分离鉴定及血清学调查

应用生化鉴定和PCR技术对1999～2001年主要分离自福建省患典型败血症鳗鲡的气单胞菌进行精细的鉴定，结果共得到气单胞菌115株，其中嗜水气单胞菌69株，温和气单胞菌29株，豚鼠气单胞菌2株和未定种气单胞菌15株。45株可被典型分型于5个主要血清型；福建鳖源嗜水气单胞菌优势血清型为O：Ah10501，鳗源气单胞菌分离株主要分布于0：Ahl0501、O：YT-1、0：CHS-3和O：CQ-1等4个血清型。非福建分离株则集中分布在O：Ah9617(0：9)。LPSs免疫印迹法既能用于不同血清型菌株脂多糖的结构分析，又能对常规血清学分型结果进行校正，其结果更直观、准确。     

欧洲鳗免疫球蛋白单克隆抗体的制备与特性

应用杂交瘤单克隆抗体技术制备了13个分泌抗欧洲鳗免疫球蛋白的单克隆抗体细胞株，并对这些单克隆抗体的特性进行了分析。经抗体级份和亚级份测定，其中IgM有3株，IgG1有5株，IgG2a有3株，IgG2b有2株；所有的单克隆抗体与欧鳗免疫球蛋白均有ELISA反应特性，抗体滴度10^4-10^7，有七株单抗具有WESTERN BLOT反应特性，能在变性条件下识别欧鳗免疫球蛋白的重链。进一步实验证实这些单抗能特异地识别欧洲鳗、日本鳗的免疫球蛋白，而与鲫、淡水白鲳、罗非鱼、胡子鲶、美洲斑点叉尾Hui血清免疫球蛋白以及水产动物常见病原菌如气单胞菌、爱德华氏菌、弧菌、柱状屈桡杆菌、沙门氏菌及大肠杆菌等无任何交叉反应。单克隆抗体9D7，对纯化的欧洲鳗免疫球蛋白的检测灵敏度为16ng。实验结果证明这些单抗具有高度特异、高度灵敏等特点，可用于鳗鲡免疫球蛋白的结构分析、免疫应答水平监测和病原诊断，具有广阔的应用前景。     

日本鳗鲡体表溃疡病病原菌的分离、鉴定及单克隆抗体制备

从日本鳗鲡(Anguilla japonica)体表溃疡病的肝脏分离到一株优势细菌JS70322NA,经人工感染证实为引起日本鳗鲡体表溃疡病的病原菌.通过形态学观察、API 20E细菌鉴定试剂盒鉴定、生理生化试验和16S rRNA特异性基因分析,鉴定该菌为嗜水气单胞菌(Aeromonas hydrophila).JS70322NA为典型的β-溶血,血清型为O∶9.以甲醛灭活的JS70322NA为抗原免疫Balb/c小鼠,利用淋巴细胞杂交瘤技术,制备出3株抗嗜水气单胞菌的单克隆抗体,分别命名为:1A5、1B8、1F3;其腹水效价分别达1.0×10-6、1.0×10-5、1.0×10-6;细胞亚型分别为IgG2a、IgM、Ig2a;3株单克隆抗体与温和气单胞菌(Aeromonas sobria)、豚鼠气单胞菌(Aeromonas caviae)、鳗弧菌(Vibrio anguillarum)等6种水产常见致病菌无交叉反应,特异强,可应用于日本鳗鲡嗜水气单胞菌特异性检测.     

耐喹诺酮类药物嗜水气单胞菌的分离鉴定及电镜观察

本研究对吉林省内12个水域采集和送检的66尾病鱼进行细菌分离鉴定,共检出46株嗜水气单胞菌疑似株,通过生化试验和PCR扩增气溶素基因aer鉴定法,确定其中22株为嗜水气单胞菌。进一步采用纸片扩散法和双倍稀释法测定嗜水气单胞菌分离株对多种抗生素的耐药性,其中部分菌株存在不同程度的耐药性,22株嗜水气单胞菌中有4株对喹诺酮类药物耐药,并且为交叉耐药。其对喹诺酮类药物的抑菌圈均小于19mm。嗜水气单胞菌耐喹诺酮类药物的检出率为18 2%。将病料中分离的8株嗜水气单胞菌在电镜下观察发现,2株耐药菌表面发现均匀分布的球形结构,而6株敏感菌表面未见此结构。     

27株嗜水气单胞菌致病性及ERIC—PCR指纹图谱分析

从福建省淡水养殖鱼类体内分离到27株嗜水气单胞菌,根据嗜水气单胞菌16SrDNA基因和气溶素基因（aerolysin）的保守序列,设计2对引物,对所分离到的27株嗜水气单胞菌进行双重PCR扩增,扩增结果表明,其中18株为含有Aer毒力基因的潜在致病性嗜水气单胞菌,占总菌数的66．67％。应用ERIC-PCR分型技术对27株嗜水气单胞菌菌株进行分析,以相似度54．00％为限,所有菌株可分为Ⅰ和Ⅱ两大聚类,以76．00％相似度为界,27株嗜水气单胞菌可分为11个聚类,同一个聚类中菌株分离区域基本相同。分析结果表明,分离的嗜水气单胞菌基因型的多样性和分离地域具有一定的关联,也表明ERIC-PCR可以有效应用于嗜水气单胞茵分子流行病学调查。     

鱼源乳酸菌对水产病原菌的抑制作用研究

为研究高活性乳酸菌的抑菌作用,采用牛津杯法体外评价了6株鱼源乳酸菌对9种水产致病菌的抑菌特性,并通过纸片扩散法测定21种抗生素对嗜水气单胞菌的抑制作用。试验结果表明,6株乳酸菌对嗜水气单胞菌、温和气单胞菌、维氏气单胞菌及豚鼠气单胞菌均有明显的抑制作用,而对鳗弧菌、大肠杆菌、弗氏柠檬酸杆菌、杀鲑气单胞菌、小肠结肠炎耶尔森菌无抑制效果,且发酵上清液的抑菌能力大于菌体。药敏试验发现仅3种药物能有效抑制嗜水气单胞菌的生长,而17种抗生素的抑菌效能已不同程度减弱甚至消失。与抗生素类药物相比,乳酸菌发酵液的抑菌能力在一定程度上优于抗生素,为抗生素替代品开发奠定基础。     

微量菌鉴系统在水产养殖中的应用

运用专门检测发酵性革兰氏阴性杆菌的微量菌鉴系统——CDRC-菌鉴系统，鉴定水产养殖中分离自患病动物血液、肝胰腺和养殖水体的9个菌株，鉴定结果为9个菌株均为弧菌科细菌，其中4株属于气单胞菌属，分别为嗜水气单胞菌、温和气单胞菌和豚鼠气单胞菌，其余5株属于弧菌属，为副溶血性弧菌和溶藻弧菌。     

鲤隐性败血症病原菌的分离与鉴定

本文对东北地区流行的鲤隐性败血症进行了病原菌的分离、人工感染、病原菌生理、生化特性和药物敏感性的测定,观察了病理组织变化等。从病鱼内脏分离到场22株致病菌,其中标7株的生长及生化特性均符合嗜水气单胞菌种的特性,嗜水气单胞菌hec毒素检测呈阳性。有3株菌V—P(-)、赖氨酸(-)、葡萄糖产气(-)是区别嗜水气单胞菌(Aeromonas hydrophila)及温和气单胞菌(A．Sobrie)主要特征,这三株菌符合豚鼠气单胞菌(A．Caviae)种的特征。     

中华鳖气单胞菌菌细胞苗和菌化学成份疫苗研制

利用分离自中华鳖的嗜水气单胞菌Ａｈ９６１００４、Ａｈ９６０９１６和Ａｈ９７０５１６与温和气单胞菌Ａｓ９７０５１０菌体及其上清制备灭活苗细胞苗和化学成份疫苗。肌肉注射中华鳖进行免疫，加强免疫１５ｄ后，用菌株Ａｈ９６１００４腹腔注射进行攻击，观察２０ｄ。结果表明，温和气单胞菌Ａｓ９７０５１０株的菌苗及其胞外分泌物（ＥＣＰ）苗组和嗜水气单胞菌Ａｈ９６１００４株的外膜成份组对嗜水气单胞菌的攻击的保护率最高     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.     

An overview of stress physiology of fish transport: changes in water quality as a function of transport duration

This study brings an integrated analysis about the relationship between water deterioration and its physiological consequences in live fish transport. The analysis was focused on the transport water and its deterioration, and physiological challenges imposed on the fish. Usual commercial handling procedures employed to mitigate fish stress during transport were discussed. Future topics of research for the establishment of safer fish transport protocols were proposed. Transport was classified into short (≤8 h) or long transport (>8 h). The main issue in short transports should be the prevention of water pH reduction, while in long transports it is the increase in ammonia. Plasma cortisol is the most employed marker for stress and is acutely elevated upon short episodes of transport, but remains elevated even in long‐transport events. Plasma glucose is perhaps a better marker for handling stress. Plasma lactate, pH, osmolality CO₂ and ions should be more often evaluated. Plasma Na⁺ and Cl^‐ are very useful markers of acidosis, due to their respective exchange for H⁺ and , for acid–base regulation. The establishment of species‐specific transport protocols should be preceded by such combined analyses of water and physiological parameters.     

Are hatchery‐reared abalone naïve of predators? Comparing the behaviours of wild and hatchery‐reared northern abalone,Haliotis kamtschatkana (Jonas, 1845)

Abalone populations have declined worldwide, generating interest in enhancement using hatchery‐reared individuals. In many cases, such restoration efforts have met with limited success due to high predator‐induced mortality rates. Furthermore, the mortality rates of outplanted hatchery abalone are often considerably higher than for wild individuals. This study uses northern abalone (Haliotis kamtschatkana) as a case study to determine whether hatchery‐reared abalone behave differently than their wild counterparts. In the field, outplanted hatchery‐reared abalone were significantly less responsive than wild abalone, in terms of number of abalone responding and intensity of response, to nearby movement and to physical contact with an inert probe. Also, when encountering a cue to which all abalone responded (a seastar predator), hatchery‐reared individuals remained subdued. Anti‐predator behavioural deficits in hatchery‐reared abalone were more pronounced in 4‐year‐old individuals than in 1‐year‐old individuals, suggesting an influence of either age or amount of time spent in the hatchery environment. These behavioural differences are expected to increase the vulnerability of hatchery‐reared abalone to predators, and are likely a major cause of their elevated predator‐induced mortality when outplanted.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd²⁺ on Ca²⁺ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l^-1 of Cd²⁺ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca²⁺ uptake within 4h resulting in a great increase in K_m values for Ca²⁺ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca²⁺ influx of larvae in solutions of 0.2 mM Ca²⁺ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd²⁺ than 0 day-old larvae. Although the Ca²⁺ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l^-1 Cd²⁺ solution, total body Ca²⁺ content was significantly reduced in 3 day-old larvae. Increased Ca²⁺ uptake efficiency ensures sufficient Ca²⁺ for normal growth. However, rapid increase in Ca²⁺ influx after hatching also leads to higher Cd²⁺ uptake. Exposure to Cd²⁺ will lead to a drop in body Ca²⁺ content resulting in retardation of larval growth. Therefore, we conclude that if Ca²⁺ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca²⁺ and will show signs of Cd²⁺ poisoning.     

Migratory dynamics of stream-spawning longnose gar (Lepisosteus osseus)

Abstract– Literature evidence suggests that lake-dwelling longnose gar (Lepisosteus osseus) enter tributary streams to spawn, Until the present study, the dynamics of this breeding migration had never been investigated quantitatively. During the summers of 1991 and 1992, longnose gar were captured as they entered Weaubleau Creek, Missouri, a tributary of Harry S. Truman Reservoir. The in-stream spawning migration began in early April and ended in late May, and was positively correlated with stream flow and water level, and negatively correlated with water temperature. In-stream residence times ranged from 15 to 94 days, with males exhibiting longer residence times than females. Once in-stream, longnose gar travelled as far as 10 km upstream and occupied certain pools at greater relative frequencies. Although the reason for this preferential utilization is not completely understood, it may relate to pool depth and riffle proximity. Longnose gar disperse from the spawning stream great distances, with gar captured in Weaubleau Creek being recaptured up to 48 km away. This information should provide fisheries biologists the means to consider the reproductive ecology of this species in their conservation and management decisions.     

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

The desaturation and elongation of [1-¹⁴C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level. This revised version was published online in August 2006 with corrections to the Cover Date.