中国本溪虹鳟一株弹状病毒的分离及初步研究

１９９０年４月,辽宁省本溪市虹鳟鱼种场的虹鳟稚鱼爆发大规模疾病,死亡率近１００％。取病鱼组织处理后接种于鱼类细胞,出现明显的细胞病变,测定细胞培养物中的病毒滴度,最高达１０￣（６．３）ＴＣＩＤ＿（５０／０．１ｍｌ）。感染病毒的细胞用１％营养琼脂糖覆盖后可形成０．５－１．５ｍｍ的蚀斑。分离到的病毒对氯仿敏感,不耐热、不耐酸,在细胞内复制的最适温度为１５℃。室内人工感染的虹鳟稚鱼能复制出与天然发病相同的症状。对感染病毒的细胞制成超薄切片,透射电镜观察,病毒长为１３１－１７３ｎｍ,平均１４８ｎｍ,直径为５８－９１ｎｍ,平均７１ｎｍ,形状为子弹状,表明是弹状病毒,有囊膜及纤突。采用挑斑法得到了纯化的病毒株,暂称之为传染性造血器官坏死症病毒中国本溪株ＩＨＮＶ－Ｂ。     

一例虹鳟低死亡率疾病的病原学及病理学研究（英文）

为确定病原类型和造成虹鳟低死亡率的原因,研究对患病虹鳟进行了病理学观察、病毒的分离和鉴定以及动物感染实验。临床检查发现发病虹鳟体色变黑,肌肉和腹壁点状出血。病理学观察发现虹鳟造血器官脾和肾间组织严重坏死。通过反转录PCR法检测坏死组织和病变细胞中传染性造血器官坏死病毒、出血性败血病毒和传染性胰腺坏死病毒,并对得到的371 bp大小片段进行测序和构建进化树分析,发现感染病原为传染性造血器官坏死病毒。同时,给体重为1.5 kg健康虹鳟腹腔注射10~4 TCID_(50)的组织滤液,累计死亡率达到35%。除此之外,将组织滤液接种到鲤鱼上皮瘤细胞系后出现了特征性病变。在实验过程中未发现细菌或寄生虫感染。结果证实引起虹鳟低死亡率的病原为传染性造血器官坏死病毒。     

点状气单胞菌引起虹鳟感染症及其病原学机制的初步研究

对山西省朔县省虹鳟试验场1987年8月在亲鱼中爆发的一次流行病进行了病原菌分离、培养、毒力试验和详细的生理生化测定,确定点状气单胞菌为其病原菌。并对该菌胞外产物的致病性进行了研究,通过测定其溶血活性、酪蛋白酶活性、对鱼致死性以及对其引起的组织病理学变化的观察,初步确定了胞外产物与该菌侵袭力的相关性。     

低鱼粉饲料中添加苏氨酸对三倍体虹鳟抗氧化能力、消化生理及肠道炎症因子基因表达的影响

研究低鱼粉饲料中不同水平苏氨酸对三倍体虹鳟(Oncorhynchus mykiss)生长、抗氧化能力、消化生理及肠道炎症因子基因表达的影响, 以初始体质量(18.42±0.20) g的三倍体虹鳟为研究对象, 在室内200 L流水水族箱中进行摄食生长试验56d。在基础饲料中添加L-苏氨酸, 配制苏氨酸水平分别为0.45%(对照)、0.76%、1.09%、1.29%和1.64%的5种等氮等能饲料。将450尾三倍体虹鳟随机分为5组, 每组处理3个重复, 每个重复30尾鱼。结果表明, 当添加量为1.09%、1.29%和1.64%时, 血清超氧化物歧化酶(SOD)活力显著提高, 同时显著降低了丙二醛(MDA)的含量(P<0.05), 过氧化氢酶(CAT)在1.29%组含量最高, 相比对照组差异显著(P<0.05)。在饲料中添加苏氨酸(0.76%—1.64%)时, 肠道脂肪酶(LPS)和胰蛋白酶的消化酶活性显著高于对照组(P<0.05), 但随着饲料苏氨酸水平达到1.64%时, 消化酶活性有所降低。1.09%和1.65%组的肠道组织形态最佳, 绒毛发达, 排列整齐, 无融合和脱落现象。苏氨酸水平对肠道炎症因子IL-2、IL-8、IL-10、IgM、TNF-α和PepT1都有显著影响(P<0.05), 随着苏氨酸水平的增加, IL-2、IgM和PepT1呈现先升高后降低的变化趋势, 在1.29%和1.09%组的表达量达到最高; IL-8呈逐渐升高趋势; 而IL-10和TNF-α呈下降的变化趋势。研究结果表明, 在低鱼粉饲料(15%)中添加适量水平的苏氨酸, 对提高三倍体虹鳟幼鱼生长、消化酶活性、促进肠道形态结构发育和增强免疫具有积极的作用。     

多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

根据基因库中对虾桃拉综合征病毒（TSV）、白斑综合征病毒（WSSV）、传染性皮下和造血器官坏死病毒（IHHNV）的基因序列,分别设计了三对特异性引物,通过对多重RT—PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT—PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp（TSV）、593bp（WSSV）和356bp（IHHNV）的特异性多重RT—PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pgTSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I—HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。     

三倍体虹鳟性转换及性腺发育的研究

用高温刺激法获得三倍体虹鳟幼鱼。从开始投饵起,分别投喂含有不同浓度17β-雌二醇(0～60μg/μg)的饵料,持续60天,获得了93%～100%的高三倍体雌性率(对照组的雌性率为52%)。三倍体雄鱼可观察到其精子形成,但性腺成熟度较相对的二倍体差;三倍体雌鱼的生殖细胞多停留在偶线期到粗线期,未发现有性成熟个体。实验结果推测,三倍体虹鳟雌鱼的不育现象看来不是由于雌雄基因组的组成差异而致,更可能是由于三组染色体造成对虹鳟卵巢发育更大的阻力。雌二醇处理对三倍体虹鳟幼鱼的生长和成活没有明显的影响。     

江苏盐城地区异育银鲫大红鳃疾病病原学研究及病理学观察

为鉴定近期江苏盐城地区养殖异育银鲫大红鳃疾病病原,研究从患病濒死鱼中分离到两株致病菌并对其理化特性、分子生物学、感染病理学进行了研究,同时开展了鉴别诊断和回归试验以及药敏检测。结果显示,两分离株理化特性与温和气单胞菌（A.sobria）特征相符,与GenBank中A.sobria的gyrB基因同源性达99%以上,在系统发育树上均与A.sobria聚为一族。同时与鲤科疱疹病毒2型（CyHV-2）鉴别诊断结果表明,患病鱼组织中CyHV-2检测为阴性。组织学上,鳃和肾组织具有典型病理变化。人工感染两株A.sobria后,鱼体出现与自然发病相似的大红鳃症状,并能从发病鱼组织中再分离到相同病原菌;而人工感染CyHV-2组织匀浆后的鲫虽然出现死亡,但无大红鳃症状出现。综上,确认此次大红鳃疾病的病原为A.sobria。药敏实验表明,分离菌株对诺氟沙星、氧氟沙星、氯霉素三种药物敏感。研究为临床上更好的预防异育银鲫大红鳃疾病奠定了基础。     

南美白对虾传染性皮下及造血组织坏死病毒抗性相关基因的微卫星标记研究

传染性皮下及造血组织坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)是南美白对虾养殖中最普遍的虾病毒之一.它能引起生长缓慢和畸形,通称矮小畸形综合症(RDS).分子标记是研究抗病基因的有效手段之一,微卫星(microsatellite)标记是新发展的遗传标记.介绍用微卫星技术对南美白对虾抗病和感病群体筛选出的IHHNV抗性相关基因进行研究.     

传染性脾肾坏死病毒(ISKNV)感染途径、宿主范围及对温度敏感性的研究

传染性脾肾坏死病毒(ISKNV)无细菌滤液通过肌肉注射、划痕浸泡、腹腔注射和口服等四种感染途径,人工感染健康鳜鱼(Siniperca chuatsi),四种途径都能引起典型的传染性脾肾坏死病毒病.通过腹腔注射感染途径,病毒滤液在25～34 ℃条件下,能引起健康鳜鱼发病.另外,用病毒滤液感染尼罗非鲫(Oreochromis niloticus)、草鱼(Ctenopharyngodon idellus)、乌鳢(Ophiocephalus argus)、大口黑鲈(Micropterus salmoides)和尖吻鲈(Lates calcarifer)五种鱼,大口黑鲈能够感染成功,为ISKNV的宿主,而其它鱼不能感染成功,不是ISKNV的宿主.     

香豆素联合传染性造血器官坏死病毒灭活疫苗对虹鳟的免疫效果

为开发安全有效的疫苗以预防虹鳟传染性造血器官坏死病毒(IHNV)暴发。研究重点开发了以香豆素为佐剂的IHNV灭活疫苗,疫苗安全性实验证明IHNV病毒液已被完全灭活。实验分为对照组、疫苗组和香豆素+疫苗组。疫苗注射剂量为150μL,对照组注射同等剂量的磷酸盐缓冲溶液(PBS),在疫苗接种14d(D14)后进行IHNV攻毒实验,并在D7、D14、D15、D21、D30进行虹鳟脾脏和血清的样本采集。IHNV攻毒后显示,香豆素+疫苗的存活率为66%,而单一灭活疫苗和对照组的分别为62%和24%。组织病理学分析显示, IHNV攻毒后香豆素+疫苗组脾脏组织损伤最小,出现轻微的炎症反应,而对照组出现严重的黑色素巨噬细胞中心(MMC)和组织炎症(TI)。此外,在D7—D30,香豆素+疫苗组血清的总超氧化物歧化酶(T-SOD)、过氧化氢酶(CAT)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)活性水平显著高于疫苗组和对照组(P<0.05)。在D7—D14,香豆素+疫苗组血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)水平被显著升高;而IHNV攻毒后(D15—D30),香豆素+疫苗组GPT和GOT水平显著低于对照组(P<0.05)。在接种疫苗和攻毒后,香豆素+疫苗组中脾脏的MHC-Ⅰ、MHC-Ⅱ和IFN-γ基因被显著上调(P<0.05);同时,香豆素+疫苗组CD4、CD8、IgT、C3、VIG1和Mx1基因表达量显著高于疫苗组和对照组(P<0.05)。结果表明,灭活疫苗联合佐剂香豆素是预防IHNV感染的有效方法,有助于进一步控制虹鳟养殖中的IHNV暴发。     

Haematological disturbances and osmotic shifts in rainbow trout,Oncorhynchus mykiss (Walbaum) under acid and aluminium exposure

Summary Elevated values of haematocrit were observed in rainbow trout (Oncorhynchus mykiss) during combined acid and aluminium exposure. Possible causes of this, i.e., decreased plasma volume, swelling of erythrocytes, and/or mobilization of erythrocytes into the blood were investigated. Slight haematocrit increases (10–20%) during mild acid stress (pH 5.0, 25 mol Ca²⁺·1^-1) were mostly caused by osmotic shifts. Both swelling of erythrocytes rocytes and a significant decrease of the plasma volume were demonstrated in fish at pH 5.0. These osmotic disturbined were greater during acid exposure (pH 5.0) combined with Al (60 g·1^-1; 200 g·1^-1). In addition, numbers of erythrocytes increased by 40% compared to acid exposure, which contributed to the severe haematocrit rise (35%) during Al exposure. A contraction of the spleen releasing erythrocytes into the blood is suggested to occur as an adrenergic response to hypoxia, which is observed in fish acutely exposed to Al.Abbreviations BV blood volume - ECFV extracellular fluid volume - ICFV intracellular fluid volume - ISFV interstitial fluid volume - MCHC mean cell haemoglobin concentration - MCV mean cell volume - PV plasma volume     

Effect of damming on distribution of rainbow trout in Hokkaido,Japan

Rainbow trout introduced into Hokkaido in 1920 have become widely distributed due to extensive release into many reservoirs and lakes for sport-fishing; their presence often results in reductions of native fish populations. We analyzed and predicted the relationship between the probability of occurrence of rainbow trout and the proximity of dams (or attributed reservoirs), using a database of the presence or absence of rainbow trout collected during 1960–2004 in Hokkaido to clarify the spread patterns of exotic species (e.g., rainbow trout) due to large-scale damming over a long period. Rainbow trout were abundant in streams within approximately 10 km of dams in recent years, regardless of whether the stream was up- or down-stream from the dam and after accounting for the effects of other environmental variables (e.g. elevation, population density, and survey year). A delayed increase in trout occurrence below dams as compared with above dams suggests that the occurrence below dams may be largely due to escapement of stocked populations and a continuously increasing abundance since 1970. The management of dams and reservoirs is necessary to prevent further spread of rainbow trout because they can threaten habitats of native Japanese salmonids through various mechanisms.

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Membrane order conservation in raft and non-raft regions of hepatocyte plasma membranes from thermally acclimated rainbow trout

Homeoviscous adaptation (HVA), the thermal conservation of membrane fluidity/order at different body temperatures, has been observed to varying degrees in different membranes. However, HVA has not been studied in raft and non-raft regions of the plasma membrane (PM) separately. Rafts are ordered PM microdomains implicated in signal transduction, membrane traffic and cholesterol homeostasis. Using infrared spectroscopy, we measured order in raft-enriched PM (raft) and raft-depleted PM (RDPM) isolated from hepatocytes of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20 °C. We found approximately 130% and 90% order compensation in raft and RDPM, respectively, suggesting their independent regulation. Raft was more ordered than RDPM in the warm-acclimated trout, a difference fully explained by a 58% enrichment of cholesterol, compared to RPDM. Unexpectedly, raft and RDPM from cold-acclimated trout did not differ in cholesterol content or order. Freezing the membrane samples during preparation had no effect on order. Treatment with cyclodextrin depleted cholesterol by 36%, 56%, and 55%, producing significant decreases in order in raft and RDPM from warm-acclimated trout and RDPM from cold-acclimated trout, respectively. However, a 69% depletion of cholesterol from raft from cold-acclimated trout had no significant effect on order. This result, and the lack of a difference in order between raft and RDPM, suggests that raft and non-raft PM in cold-acclimated trout are not spatially segregated by phase separation due to cholesterol.     

Serotonin, but not melatonin, plays a role in shaping dominant-subordinate relationships and aggression in rainbow trout

The aim of this study was to clarify to what extent the effects of elevated dietary L-tryptophan (Trp) on aggressive behavior and stress responsiveness in rainbow trout are mediated by circulating melatonin and central serotonin (5-HT), respectively. Isolated rainbow trout were paired for 1h a day for 7 days in order to create fish with experience of being dominant and subordinate. Following this week, the fish were tested for aggressive behavior using a resident-intruder test after which they were subjected to one of four treatments: (1) tryptophan, (2) the selective serotonin reuptake inhibitor (SSRI) citalopram, (3) melatonin, and (4) no treatment (controls). After 7 days of treatment, the fish were subjected to a second resident-intruder test. Trp-supplemented feed resulted in a suppression of aggressive behavior in fish with experience of being dominant. Moreover, fish fed Trp-supplemented feed, regardless of social experience, also displayed lower plasma cortisol levels than controls. These effects of elevated dietary Trp were closely mimicked by citalopram treatment, whereas exogenous melatonin had no effect on either aggressive behavior or plasma cortisol. Thus, the effect of elevated dietary Trp on aggressive behavior and stress responses does not appear to be mediated by melatonin even though elevated dietary intake of Trp resulted in an increase in plasma melatonin concentrations.