An analysis of levels of infectious pancreatic necrosis virus in Atlantic salmon,Salmo salar L., broodstock in Scotland between 1990–2002

Throughout this study period the prevalence of infectious pancreatic necrosis virus (IPNV) in Scottish farmed Atlantic salmon was high in the marine environment but relatively low in fresh water. In order to minimize the risk of vertical transmission of infection from parent to progeny, all IPNV infected broodstock populations had to undergo testing of all fish for the virus at the time of stripping and eggs from positive parents were destroyed. Between 1990 and 2002 over 68 000 Atlantic salmon broodfish were individually screened for IPNV by cell culture isolation and enzyme linked immunosorbent assay. Generalized linear mixed models were used to assess the influence of geographical region, age, sex and year on IPNV prevalence in Atlantic salmon broodstock. This analysis determined that the age and sex of the broodfish and the geographical region of the broodstock stripping site did not have a statistically significant influence on IPNV prevalence within the broodstock parental population. However, there was a statistically significant temporal increase in IPNV prevalence from 1990 to 2002.     

Butenolides from the Coral-Derived Fungus Aspergillius terreus SCSIO41404

Five undescribed butenolides including two pairs of enantiomers, (+)-asperteretal G (1a), (−)-asperteretal G (1b), (+)-asperteretal H (2a), (−)-asperteretal H (2b), asperteretal I (3), and para-hydroxybenzaldehyde derivative, (S)-3-(2,3-dihydroxy-3-methylbutyl)-4-hydroxybenzaldehyde (14), were isolated together with ten previously reported butenolides 4–13, from the coral-derived fungus Aspergillus terreus SCSIO41404. Enantiomers 1a/1b and 2a/2b were successfully purified by high performance liquid chromatography (HPLC) using a chiral column, and the enantiomers 1a and 1b were new natural products. Structures of the unreported compounds, including the absolute configurations, were elucidated by NMR and MS data, optical rotation, experimental and calculated electronic circular dichroism, induced circular dichroism, and X-ray crystal data. The isolated butenolides were evaluated for antibacterial, cytotoxic, and enzyme inhibitory activities. Compounds 7 and 12 displayed weak antibacterial activity, against Enterococcus faecalis (IC₅₀ = 25 μg/mL) and Klebsiella pneumoniae (IC₅₀ = 50 μg/mL), respectively, whereas 6 showed weak inhibitory effect on acetylcholinesterase. Nevertheless, most of the butenolides showed inhibition against pancreatic lipase (PL) with an inhibition rate of 21.2–73.0% at a concentration of 50 μg/mL.     

茯砖茶不同萃取物对消化酶活性的影响

将茯砖茶依次用三氯甲烷、乙酸乙酯及正丁醇萃取后制备样品,测定各部分样品对α-淀粉酶及胰脂肪酶活性的影响。研究发现茯砖茶各部分样品对胰脂肪酶活性的影响程度不同,三氯甲烷层稍有抑制作用,活性只有对照的0.82倍,水提物、乙酸乙酯萃取层及正丁醇萃取层有显著的激活作用,正丁醇萃取层部分激活作用最明显,达2.54倍。茯砖茶各部分样品对α-淀粉酶活性均有激活作用,其中以乙酸乙酯萃取层部位的激活作用最强,激活倍数为4.09,其次是水提物,其激活倍数为2.99,水层的激活作用不显著。三氯甲烷萃取层抑制作用不显著,活性只有对照的0.88倍。乙酸乙酯萃取层及正丁醇萃取层部分经过HP-20树脂再分离后,各部分仍有较高的活性。     

Genetic analysis of infectious pancreatic necrosis virus from Scotland

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish, and is one of the most serious economic diseases in aquaculture. In Scotland, an increase in IPN virus (IPNV) outbreaks in seawater Atlantic salmon, Salmo salar, has been reported in recent years. The aim of this study was to analyse the VP2 gene from recent IPNV isolates from Scotland, to determine whether there are epidemiological links between IPNV isolates from farms (13), wild fish (17) and the environment (6) in order to investigate potential wild and farmed fish interactions. Comparison of the nucleotide sequence of the VP2 gene revealed that 34 of 36 isolates were 97.1-100% similar and the deduced amino acid sequences showed 97-100% identity. Two isolates from wild fish exhibited the most divergence at 85-87.3% similarity to the other isolates at the nucleotide level and 88.2-90.8% identity at the deduced amino acid level. Phylogenetic analyses revealed that 34 of 36 of the isolates from Scotland were genetically closely related to the A2 (Sp) serotype of IPNV. The two wild isolates from seatrout, Salmo trutta, and flounder, Platichthys flesus, were most closely related to the European A5 (Te) serotype. This study represents a comprehensive IPNV phylogenetic study that indicates that there are closely related or identical isolates in circulation in the marine environment, which adds evidence that disease interactions between wild and farmed fish may occur. This type of analysis is a useful tool in the management and control of fish diseases because it can assist in the identification of epidemiological links and highlight potential risks to aquaculture.     

Estimation of infectious dose and viral shedding rates for infectious pancreatic necrosis virus in Atlantic salmon,Salmo salar L., post‐smolts

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post‐smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10² to 10^?4 50% end‐point tissue culture infectious dose (TCID₅₀) mL^?1 sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post‐smolts was <10^?1 TCID₅₀ mL^?1 by bath immersion (4 h at 10 °C). The peak shedding rate for IPNV following intraperitoneal challenge using post‐smolts was estimated to be 6.8 × 10³ TCID₅₀ h^?1 kg^?1 and occurred 11 days post‐challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.     

An improved in situ hybridization method for the detection of fish pathogens

A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.     

牙鲆生长抑素分泌细胞的免疫细胞化学研究

采用链霉亲合素-生物素-过氧化物酶复合物(SABC)免疫细胞化学染色技术,利用兔抗哺乳动物生长抑素血清对牙鲆(Paralichthys olivaceus)消化道(包括食道、胃、幽门垂、肠道全长及肛门)、肝胰脏、肾脏、脾脏以及生殖腺等器官的生长抑素(SS)分泌细胞进行定位和鉴别。结果表明,生长抑素分泌细胞仅存在于胃和胰岛中,其他检测部位均未见分布。在胃中,生长抑素分泌细胞主要散在分布于胃黏膜上皮、胃小凹底部上皮和胃腺的颈部周围。在胃的不同部位,生长抑素分泌细胞的分布密度存有差异,以胃体部数量最多,贲门部次之,幽门部最少。参照消化道内分泌细胞与胃肠腔或腺腔有无直接联系,以及基部有无胞质突起的分类方法,将SS分泌细胞分为4种不同的类型,这4种类型的SS分泌细胞在胃中均有出现,且不同部位出现的几率不同。其中,贲门部以第Ⅲ、Ⅰ型居多;胃体部以第Ⅳ、Ⅰ型细胞为主;幽门部仅有第Ⅰ、Ⅱ型细胞。提示,牙鲆的胃是一个复杂的内分泌器官。在胰脏中,生长抑素分泌细胞零散地分布于胰岛细胞之间,数量很少。     

罗汉果皂苷提取物对INS-1胰岛β细胞糖脂毒性的保护作用研究

为阐明罗汉果皂苷提取物(MGE)对胰岛β细胞糖脂毒性的保护作用,本试验通过噻唑蓝(MTT)比色法、Hoechst 33342/PI荧光染色法和Western blotting研究MGE对高糖、高脂诱导INS-1胰岛β细胞活力降低、凋亡及促凋亡因子caspase 3表达水平的影响。结果表明,在0.1~100 μg·mL^-1浓度范围内,MGE对INS-1细胞无明显细胞毒性(P>0.05)。高糖、高脂处理使INS-1胰岛β细胞活力显著降低(P<0.05),而MGE(0.1~100 μg·mL^-1)干预可明显增强INS-1细胞活力,并呈一定的剂量效应关系。与高糖高脂模型组相比,100 μg·mL^-1MGE干预使INS-1胰岛β细胞活性上升了15.5%。荧光染色表明,MGE处理明显抑制了高糖、高脂诱导的INS-1胰岛β细胞凋亡。免疫印迹结果表明,高糖、高脂处理的INS-1胰岛β细胞,cleaved caspase 3蛋白表达水平显著上调,而MGE干预显著下调cleaved caspase 3水平(P<0.05)。本研究结果表明,MGE干预改善了胰岛β细胞的糖脂毒性,抑制了细胞凋亡,其抗凋亡作用可能与抑制caspase 3的剪切活化有关。本研究结果为罗汉果高附加值产品的开发及天然、安全降糖功能因子的筛选提供了一定的理论依据。     

鹅胰液的分泌及其调节研究

选成年杂交 (川白×太湖 )鹅 8只 ,用胰腺—十二指肠长久性回流瘘管手术 ,在不限制鹅运动和采食情况下收集纯净的胰液。实验期饲以大麦 (占 45 % )基础日粮 ,白天 (8:0 0～ 2 0 :0 0 )给食 ,夜间 (2 0 :0 0～ 8:0 0 )禁食 ,自由饮水。实验期连续收集胰液。结果表明 :鹅的胰液呈不规则的连续性分泌。其分泌速率白天为 (30 7 5 9± 2 9 40 ) μL/h ,夜间为 (10 0 0 0± 2 2 45 ) μL/h ;分泌量白天占 73 6 6 % ,夜间占 2 6 34%。皮下注射硫酸阿托品 0 5mg/只 ,则分泌速率分别降至对照期的 2 4 0 0 % (白天 )和 2 8 77% (夜间 ) (P <0 0 1) ;胰液蛋白酶、脂肪酶和淀粉酶的活力也较对照期下降(P <0 0 1)。表明鹅的胰液分泌受胆碱能神经调节。口服丙谷胺 15 0mg/kg ,胰液分泌速率试验期与对照期差异不显著(P >0 0 5 ) ;而胰液蛋白酶、脂肪酶和淀粉酶的活力显著下降 (P <0 0 1)。表明丙谷胺通过阻断CCK受体而影响胰酶的分泌 ,但对胰液分泌量影响不大     

一株虹鳟源传染性胰腺坏死病病毒的分离与鉴定

为明确引起四川石棉某养殖场饲养的虹鳟患病死亡的病原体,实验对自然发病虹鳟进行大体病变观察并对其病原体进行分离,通过人工感染实验及多重RT-PCR鉴定确定病原体WZ160509,并对病原体的主要结构蛋白VP2进行扩增分析,同时对病变组织进行组织病理学观察。结果显示,患病鱼主要临床症状表现为体表发黑,腹部膨大,挤压腹部可见肛门喷射淡黄色黏液便;剖检可见肝脏、肾脏苍白;肠道内无食物,内积黄色黏液。将患病虹鳟组织匀浆液无菌接种虹鳟鱼生殖腺细胞系(rainbow trout gonad cell line,RTG-2)细胞,盲传3代均出现典型的细胞病变。人工感染实验显示死亡率高达90%,并出现与自然患病鱼相同的症状。多重RT-PCR检测发现,自然发病鱼、人工感染鱼以及病变RTG-2细胞均为传染性胰腺坏死病病毒(infectious pancreatic necrosis virus,IPNV)阳性,其主要结构蛋白VP2基因与美国分离株基因组1型聚为一支,且同源性分析表明,WZ160509-VP2与IPNV-VP2(AY026345)的同源性最高,序列一致性为95.8%。组织病理学观察显示,患病鱼胰腺细胞空泡变性,坏死;肝细胞空泡变性,坏死;肾小球轻度炎症,毛细血管通透性增加,肾小囊腔内有红色絮状蛋白类物质渗出,肾小管上皮细胞空泡变性。研究表明,从该养殖场患病虹鳟中分离到的病毒为IPNV。