乌梅-全蝎双药方对气单胞菌混合感染大口黑鲈的治疗效果分析

【目的】了解和掌握嗜水气单胞菌(Aeromonas hydrophila,Ah)、温和气单胞菌(Aeromonas sobria,As)、维氏气单胞菌(Aeromonas veronii,Av)混合感染大口黑鲈的致病性并筛选出防治气单胞菌感染的有效中药,为气单胞菌混合感染的防治提供理论依据。【方法】将Ah、As和Av通过腹腔注射健康大口黑鲈,构建3种气单胞菌单独感染和混合感染大口黑鲈的动物模型,通过脏器系数、组织病理切片和死亡率等分析气单胞菌混合感染的致病性;通过体外抑菌试验从37味中药中筛选对气单胞菌具有抑制效果的敏感中药,利用网络药理学解析乌梅-全蝎双药方在体内发挥抗菌作用的关键活性成分和作用靶点,并通过药浴试验分析其对气单胞菌混合感染大口黑鲈的治疗效果。【结果】3种气单胞菌混合感染对大口黑鲈的致病性高于单独感染,Ah能造成最严重的脏器损伤、炎症和最高的死亡率,As次之,Av最弱。中药乌梅和全蝎对Ah、As和Av在体外均具有显著抑制效果,二者联用时抑菌效果增强。网络药理学分析发现,乌梅-全蝎双药方的活性成分和细菌感染的交集作用靶点共180个,其中肿瘤蛋白53(tumor prote...     

大口黑鲈和中华绒螯蟹心脏组织结构及ATP酶含量的比较

研究比较了大口黑鲈(Micropterus salmoides)和中华绒螯蟹(Eriocheir sinensis H.Milne Edwards)心脏组织结构、肥大细胞和ATP酶含量.结果发现,大口黑鲈与中华绒螯蟹心脏组织结构明显不同.中华绒螯蟹心肌纤维排列更加松散,线粒体形态多样,嵴短而松散;大口黑鲈的心肌纤维有明显的明带和暗带,而中华绒螯蟹的仅见长度约为1.5 μm的暗带;大口黑鲈与中华绒螯蟹心肌组织间肥大细胞着色特性、形态及分布相似,中华绒螯蟹心脏组织中肥大细胞长径比大口黑鲈的略大,长约为20.6μm.透射电镜下观察,肥大细胞胞浆中充满高电子密度颗粒.在中华绒螯蟹心室中发现有与肥大细胞形态相似的白细胞.中华绒螯蟹心脏指数显著高于大口黑鲈,但大口黑鲈心肌组织ATP含量远高于中华绒螯蟹.结果表明大口黑鲈的心脏组织结构比中华绒螯蟹更为复杂,两种动物心肌间均发现有肥大细胞的分布,中华绒螯蟹单位心脏重量所含ATP酶的量明显比大口黑鲈低,这可能与它为开放式循环供能所需能量较低有关.     

大口黑鲈主要病害研究进展

大口黑鲈(Largemouth bass,Micropterus salmoides)是我国主要的水产养殖品种,近年来病害多发,导致养殖业损失严重。本文从细菌性疾病、病毒性疾病和寄生虫病三方面,概述了近年来大口黑鲈总体病害情况,描述了疫病的病原特征、发病时间和主要症状,并从机体分子机制、疫苗研究、防控方法等方面,总结了国内外的最新研究进展,指出未来的病害防控方向可从疫苗和中草药制剂等方面着手,研发新型、高效、安全的绿色渔药或疫苗。     

一株鲈塘源植物乳杆菌的分离鉴定及其对养殖水体影响研究

利用MRS培养基和LB培养基对广东顺德大口黑鲈养殖池塘水体样品进行了乳酸菌的分离和筛选,共获得27株乳酸菌。以引起大口黑鲈"烂身病"的嗜水气单胞菌为指示菌进行拮抗作用试验,筛选获得一株具有显著抑菌活性的乳酸菌XTL3,并对该菌株进行了生理生化试验、16Sr DNA序列分析及其对大口黑鲈池塘养殖的安全性和效果进行了研究。结果表明,菌株XTL3对嗜水气单胞菌的生长具有较好抑制作用。生理生化试验、16S r DNA序列分析表明,XTL3与植物乳杆菌的相似性为100%,初步鉴定该菌株为植物乳杆菌(Lactobacillusplantarum)。室外养殖池塘安全性和效力检验结果表明,在使用过程中的一个月内,无异常死亡,无病害发生,池塘的溶氧变化差异不显著(P0.05),氨氮的变化差异显著(P0.05),实验塘的水环境保持更稳定。筛选的植物乳杆菌具有良好的抑菌和调水能力,且其生长特性适应大口黑鲈的池塘养殖环境,这为大口黑鲈疾病的生态防控及乳酸菌资源的开发利用奠定了基础。     

大口黑鲈源豚鼠气单胞菌的分离鉴定及全基因组分析

【目的】探究患病大口黑鲈的致病菌及其主要生物学特性,为临床疾病防控提供技术支撑。【方法】首先对发病大口黑鲈进行病理组织学观察,进而利用平板划线法分离细菌,通过革兰染色、生化试验和16S rDNA序列测定对分离菌进行鉴定,通过致病性试验观察分离菌对小鼠主要脏器造成的病理损伤;而后分别利用PCR方法和Kirby-Bauer(K-B)纸片法对分离菌的常见毒力基因和耐药特征进行检测分析;最后利用二代测序技术绘制其全基因组草图,并通过生物信息学方法对其分子特征进行解析。【结果】自然感染大口黑鲈肝细胞肿大,空泡变性,肠黏膜下层出血,肠壁坏死;于患病黑鲈体内分离到1株优势菌,在血平板上呈乳白色,半透明,直径约1 mm的小菌落,呈β溶血,革兰染色为阴性短杆菌,两端钝圆,经生化试验和16S rDNA测序证实分离菌为豚鼠气单胞菌(Aeromonas caviae),将其命名为SCMS。试验小鼠接种后24 h内死亡率为100%,病理特征与自然感染大口黑鲈相似;SCMS株中共检测到9种毒力基因,分别为Aha、Ahp、Aer、Alt、gcaT、Act、Exu、Ela和eprCAI;药敏试验结果显示,分离菌对大多...     

大口黑鲈不同组织中抗菌肽粗提及抑菌活性测定

研究了大口黑鲈不同组织中抗菌肽的提取及其粗提物对不同菌种的抑菌作用。以新鲜的大口黑鲈为试验材料,用5％乙酸作为提取液,对大口黑鲈粘液、皮肤、肝脏、脾、卵、鳃组织进行提取,并对粗提物进行抑菌活性检测。结果表明大口黑鲈粘液、皮肤和肝脏组织提取物对嗜水气单胞菌有抑菌作用,皮肤和肝脏组织提取物对大肠杆菌有抑菌作用。肝脏组织抗菌肽提取物经SephadexG-25凝胶过滤层析柱分离后,其收集液对嗜水气单胞菌仍具有抑菌作用。大口黑鲈皮肤和肝脏组织提取物对嗜水气单胞菌、大肠杆菌均有抑菌作用。     

硫辛酸对氨氮胁迫下大口黑鲈抗氧化能力的影响

试验探析硫辛酸对大口黑鲈抗氨氮胁迫能力的影响.驯养后的大口黑鲈幼鱼分为2组,对照组为基础饵料,处理组拌喂400mg/kg的硫辛酸,每组3个重复.饲养20 d,对所有鱼进行氨氮胁迫.胁迫后6、12、24、48 h采集大口黑鲈的血清和肝脏样品,分析总超氧化物歧化酶(T-SOD)和过氧化氢酶(CAT)的活性及丙二醛(MDA)...     

冰鲜杂鱼和人工配合饲料对大口黑鲈肌肉品质及健康状况影响的评价

本试验通过测定血清生化指标和肌肉营养成分,评价冰鲜杂鱼和人工配合饲料对大口黑鲈肌肉品质及健康状况的影响。养殖过程中冰鲜组大口黑鲈全程投喂冰鲜杂鱼,饲料组大口黑鲈全程投喂大口黑鲈专用人工配合饲料。在商品鱼上市前,冰鲜组和饲料组各随机采集6尾用于测定血清生化指标,再各随机采集6尾用于测定肌肉营养成分。结果显示:饲料组大口黑鲈血清中谷草转氨酶(AST)活性显著高于冰鲜组(0.01≤P0.05),血清中碱性磷酸酶(ALP)活性极显著高于冰鲜组(P0.01),血清中总蛋白(TP)和白蛋白(ALB)含量极显著低于冰鲜组(P0.01)。冰鲜组大口黑鲈肌肉中总氨基酸(TAA)含量显著高于饲料组(0.01≤P0.05),且肌肉中各种氨基酸的氨基酸评分(AAS)和化学评分(CS)以及必需氨基酸指数(EAAI)均高于饲料组。饲料组大口黑鲈肌肉中亚油酸(C18∶2)含量极显著高于冰鲜组(P0.01)。结果表明,本试验条件下,冰鲜组大口黑鲈肌肉营养品质特别是蛋白质品质及氨基酸组成要优于饲料组,且健康状况也优于饲料组,但饲料组大口黑鲈肌肉中亚油酸含量较冰鲜组极显著升高。因此,大口黑鲈的人工配合饲料配方还需要进一步的优化,来满足消费者对大口黑鲈的营养需求。     

大口黑鲈的营养需求研究进展

大口黑鲈（Micropterussalmoides）,俗称加州鲈,原产于美国加利福尼亚州,隶属鲈形目（Perci—formes）太阳鱼科（Ceutrarchidae）。大口黑鲈于1894年引入我国,由于其具有生长快、病害少、耐低温、肉多刺少、味道鲜美、营养丰富等优点,已成为我国养殖的主要淡水鱼品种之一。目前,我国大口黑鲈的年产量已达16万t,大口黑鲈养殖区域主要分布在广东顺德和南海、江苏吴江和南京、浙江湖州、四川等地。广东大口黑鲈养殖面积已达2600hm2以上,产量占全国的759／6,位列全国第一。     

氯化胆碱使用量对大口黑鲈生长、饲料利用及形体指标的影响

以商业配方为基础,研究不同水平的氯化胆碱添加对大口黑鲈生长、饲料利用及形体指标的影响.试验使用不同梯度氯化胆碱(0,0.3％,0.6％)制作膨化饲料在池塘网箱中饲喂大口黑鲈(44.87±0.15g)60天.结果表明对于40-160g大口黑鲈,添加0.3％和0.6％的氯化胆碱组加州鲈增重率和特定生长率显著提高(P＜0.0...     

The Lethal Dose of Largemouth Bass Virus in Juvenile Largemouth Bass and the Comparative Susceptibility of Striped Bass

Abstract

Largemouth bass virus (LMBV) is an iridovirus that was isolated from wild adult largemouth bass Micropterus salmoides in the southeastern United States in 1994. Although originally isolated from moribund wild fish, its virulence to juvenile largemouth bass is uncertain. To help clarify this point, two LMBV titrations were made in juvenile largemouth bass. Titers of LMBV in fathead minnow cells were 10^4.8 and 10^5.8 tissue culture infectious doses—50% cytopathic endpoint (TCID50) per milliliter, respectively. Tenfold serial dilutions of LMBV employed in each cell culture titration, injected intraperitoneally (0.1 mL/fish) into largemouth bass produced calculated lethal dose—50% mortality endpoints (LD50s) of 282 (10^2.45) and 288 (10^2.46) infectious doses in two consecutive infectivity trials. Virus yield of assayed infected fish averaged 10^8.5 TCID50/g and 10^7.7 TCID50/g in viscera of moribund and dead fish in the two trials and 10^6.5 TCID50/g in surviving exposed fish 14 d after infection. In a second experiment, largemouth bass had 100% mortality 5 d after injection while virus immersed fish had a significantly (P ≤ 0.005) lower mortality of 17% at 14 d. Similarly treated juvenile striped bass Morone saxatilis suffered 63% mortality after injection and significantly (P ≤ 0.005) lower mortality of 10% after immersion. In a third study of 25 d, 100% of injected largemouth bass died by 5 d after injection, and all of them were virus-positive. Injected striped bass had a significantly (P ≤ 0.005) lower mortality of 24%; all three fish were virus-positive initially, two fish were virus-positive at 18 d, and none were positive at 25 d. Juvenile largemouth bass were highly susceptible to LMBV injection and striped bass were moderately susceptible, but both species were only mildly susceptible when exposed by immersion.     

Movement behaviour of largemouth bass Micropterus salmoides in a South African impoundment

The objectives of this study were to describe movements and area use patterns of largemouth bass Micropterus salmoides in relation to abiotic factors (water temperature, river inflow and water level). The study was conducted in the Kubusi arm of Wriggleswade Dam, Eastern Cape, South Africa where nine largemouth bass (310–385?mm FL) were surgically implanted with individually coded acoustic transmitters and monitored for nine months (May 2010 to January 2011) using an array of five moored acoustic data-logging receivers. The daily position occupied by largemouth bass was significantly affected by water temperature (P<0.001) and river inflow (P<0.001), whereas water level had no effect. Largemouth bass spent little or no time in the shallow river inlet areas when water temperature was <12.5 °C, but as water temperature increased, their utilisation of shallow areas increased significantly. The results from this study demonstrate the importance of environmental factors, particularly water temperature, on the movement of largemouth bass.     

Effects of Practices Related to Catch-and-Release Angling on Mortality and Viral Transmission in Juvenile Largemouth Bass Infected with Largemouth Bass Virus

Abstract

Largemouth bass virus (LMBV; family Iridoviridae) has recently emerged as a causative agent in fish kills of largemouth bass Micropterus salmoides. Little is known about how the virus is transmitted or what factors predispose fish to mortality subsequent to infection. Concern has nevertheless arisen that activities related to recreational angling may affect transmission dynamics and may alter the susceptibility of infected fish to clinical disease. This study examined the separate effects of two angling-related factors on the susceptibility of juvenile largemouth bass to mortality from LMBV infection and on the transmission of LMBV from infected to uninfected fish. The first factor was hook-and-line angling. Infected fish that underwent a simulated angling treatment did not experience higher mortality or have higher viral loads in their tissues than those that were not angled. The second factor was direct contact between infected and uninfected fish, as would occur in live wells and holding tanks. The LMBV was transmitted from infected to uninfected fish through water, even when direct contact was prevented. Transmission of LMBV between infected and uninfected fish separated by a fenestrated barrier was nearly as efficient as LMBV transmission between infected and uninfected fish that were allowed direct contact. These results imply that angling itself may have only minimal effects on the survival of largemouth bass infected with LMBV but that angling-related practices that place infected and uninfected fish together in a limited water volume may facilitate viral transmission. Partitioning or cooling of live wells and holding tanks, as well as limiting their use, could reduce LMBV-associated mortality and viral transmission.     

Survival of Largemouth Bass Iridovirus in Frozen Fish

Abstract

Juvenile largemouth bass Micropterus salmoides were infected with largemouth bass iridovirus by injection and frozen at ?10°C, and the visceral organs and tissues (liver, kidney, spleen, gonads, swim bladder, and muscle) of individual fish were periodically assayed for virus for 155 d postfreezing. The virus was isolated from 35 of 39 (89.7%) of the frozen fish. However, virus loss occurred more rapidly in fish that were dead when frozen than in fish that were alive when frozen. Overall, largemouth bass iridovirus was stable in frozen tissue when the fish were fresh upon freezing.     

Isolation of Acholeplasma laidlawii from Centrarchids in a Central Florida Lake

Abstract

In 1991, the poor physical condition of largemouth bass Micropterus salmoides from Lake Harris, Florida, was associated with the decline of the lake's fishery. The swim bladders of emaciated bass had mild inflammation and ecchymotic hemorrhages. A mycoplasma-like organism isolated from swim bladders was initially believed to be the causative agent. The organism was later identified as Acholeplasma laidlawii by using a fluorescent antibody procedure and was demonstrated to be nonpathogenic. Parenteral injection of the organism into healthy largemouth bass fingerlings produced no signs of disease or difference in growth rate compared with control fish during a 16-month period. Field studies resulted in isolation of A. laidlawii from black crappies Pomoxis nigromaculatus, bluegills Lepomis macrochirus, and redear sunfish L. microlophus, but not from noncentrarchids in Lake Harris or from any fish species in a control fishery (Lake Holly, Florida). The absence of organisms in all emaciated bass, our inability to reproduce the disease, and isolation of the organism from seemingly healthy fish suggest this organism was not pathogenic.     

A survey to determine the presence and distribution of largemouth bass virus in wild freshwater bass in New York State

During 2004 and 2005 a survey was conducted to investigate the presence and geographic distribution of largemouth bass virus (LMBV) in New York State. This iridovirus is widely distributed across the eastern United States; however, it had not previously been reported in New York State. Two hundred and eighty-three wild largemouth bass Micropterus salmoides and 8 smallmouth bass M. dolomieu were collected from 37 locations across the state. No clinical signs of LMBV or mortalities attributable to the virus were observed in the fish collected. Using a quantitative polymerase chain reaction (QPCR) method, we detected LMBV in 28 fish from 13 locations. Viral cytopathic effect in cell culture was observed in 5 fish from 3 locations. The virus isolated from cell culture was confirmed to be LMBV by an independent PCR method. Statistical analysis of the largemouth bass samples collected during 2005 revealed a wide difference in prevalence between the QPCR results and the cell culture results. Analysis of possible predictors, including age, sex, and month collected, showed no significant associations with the QPCR results. This survey confirms the presence and wide distribution of a potentially pathogenic form of LMBV in multiple water systems across New York State.     

Isolation and Identification of Viruses from Adult Largemouth Bass during a 1997–1998 Survey in the Southeastern United States

Abstract

A total of 452 largemouth bass Micropterus salmoides (mostly adults) from 78 reservoirs, lakes, rivers, and state fish hatcheries in eight states of the southeastern USA were surveyed for largemouth bass virus (LMBV) between August 1997 and November 1998. Virus was isolated from ostensibly healthy electrofished largemouth bass from six reservoirs on four different river systems—Lakes Jordan (Coosa River) and Wilson (Tennessee River) in Alabama; Lakes Walter F. George and Oliver (Chattahoochee River), Lake Blackshear (Flint River), and Lake Allatoona (Coosa River) in Georgia—and from moribund fish from Lake Walter F. George and Lake Greenwood (Santee River) in South Carolina. Fish samples from 71 locations in Alabama, Florida, Georgia, Kentucky, Louisiana, Missouri, South Carolina and Virginia showed no evidence of virus infection. Sequence analysis of the major capsid protein gene showed that virus isolates from Lakes Walter F. George, Jordan, Blackshear, and Allatoona were identical to the original LMBV isolate from Santee–Cooper Reservoir. Largemouth bass virus is a member of the genus Ranavirus within the family Iridoviridae and is nearly identical to doctor fish virus (DFV-16) and guppy virus (GV6), two fish viruses from Southeast Asia.     

Cardiac tamponade in largemouth bass (Micropterus salmoides)

A public aquarium with a 4-mo history of occasional fish mortalities submitted for necropsy an adult female largemouth bass (Micropterus salmoides) that died unexpectedly. Gross necropsy revealed that the pericardial cavity was markedly distended with partially coagulated blood. Examination of the heart revealed multiple nodular masses in the area of the atrium and two small perforations on the surface of one of the nodular masses. Histopathologic exam of the atrium revealed severe fibrinonecrotic endocarditis and transmural myocarditis with intralesional bacteria. A pure culture of Edwardsiella tarda was obtained from culture of posterior kidney and spleen. An area of stagnant water that may serve as the source of E. tarda was identified, and steps to rectify this problem were taken. Low-level supersaturation was also a significant stressor; the source of the supersaturation was not identified. To our knowledge, this is the first report of cardiac tamponade in a largemouth bass.     

大口黑鲈对饲料中酵母硒的耐受性研究

本试验旨在通过研究酵母硒对大口黑鲈生长性能、血浆生化指标、组织抗氧化指标及肝脏组织结构的影响,评价大口黑鲈对饲料中酵母硒的耐受性。在基础饲料中分别添加0(Y0)、0.5(Y0.5)、2.5(Y2.5)、5.0 mg/kg(Y5.0)(以硒计)酵母硒,其中0.5 mg/kg是硒的最高推荐剂量,2.5和5.0 mg/kg分别是最高推荐剂量(0.5 mg/kg)的5和10倍。基础饲料本底硒含量为0.76 mg/kg。选用初始体重为(12.99±0.01)g大口黑鲈,随机分为4组,每组6个重复,每个重复20尾,试验期为10周。结果表明:Y0组增重率和摄食率最低,同时其饲料系数也最低,均显著低于其余各组(P0.05)。Y0组血浆中碱性磷酸酶活性显著高于其余各组(P0.05)。Y0.5组血浆中高密度脂蛋白胆固醇含量显著高于其余各组(P0.05)。Y2.5组血浆中尿素氮含量显著高于其余各组(P0.05)。Y2.5组、Y5.0组血浆中免疫球蛋白M含量显著高于Y0组、Y0.5组(P0.05)。与Y0组相比,酵母硒的添加显著降低了血浆中丙二醛的含量(P0.05),且显著提高了血浆中谷胱甘肽过氧化物酶的活性(P0.05)。Y5.0组肝脏硒含量显著高于Y0组、Y 0.5组(P0.05),与Y2.5组无显著差异(P0.05)。硒日摄入量和肝脏硒含量呈显著线性相关(P0.05),肝脏硒含量随硒日摄入量的提高呈线性增加。各组大口黑鲈的肝脏都有不同程度的损伤,但添加0.5 mg/kg酵母硒对肝脏损伤有缓减作用。由上述结果可知,饲料中添加0.5 mg/kg酵母硒(总硒含量为1.29 mg/kg)对大口黑鲈具有一定的脂肪代谢促进作用和抗氧化保护功能,且对大口黑鲈是安全的。本试验条件下,综合生长性能、血浆生化指标、组织抗氧化指标及肝脏组织结构,饲料本底硒含量为0.76 mg/kg时,大口黑鲈对饲料中酵母硒的耐受剂量为0.5 mg/kg(以硒计),即为硒的最高推荐剂量,安全系数为1。鱼粉、磷虾粉等动物蛋白质源中含有较高水平的硒元素,因此在高鱼粉水产动物饲料中补充硒要慎重。     

Laboratory transmission of the monogenean Acolpenteron ureteroecetes infecting the posterior kidneys of largemouth bass: time course and pathology

Acolpenteron ureteroecetes infections in the kidneys of largemouth bass Micropterus salmoides have been reported, but the time course of infection and progression of pathology in experimentally infected fish remain unknown. We exposed 299 naive juvenile largemouth bass at 19.8 degrees C to A. ureteroecetes-infected largemouth bass via recirculating water without direct contact. For 7 months, prevalence and density were determined monthly in squashes of posterior kidney (20 exposed fish, 20 nonexposed fish), and histopathology was assessed in 5 fish from each group. Prevalence increased steadily from months 1 (5%) to 4 (85%), thereafter remaining relatively stable. Mean density of infection doubled monthly (month 1, 0.1 individuals/2 cm2 squash; month 7, 15.1 individuals/2 cm2 squash). Eggs were first observed at month 3, and mean density increased markedly from month 4 to month 5 (2.9-15.3 eggs/2 cm2 squash). Histopathology showed damage in renal collecting ducts that got progressively worse between 5 and 7 months. The infected ducts were dilated, had a hyperplastic epithelium, and were surrounded by chronic inflammation, including eosinophilic granular cells and varying degrees of fibrosis. Eggs within granulomas were present in the interstitium; this response is newly reported. The infection system developed in this study provides a reproducible and consistent source of infected individuals that will facilitate further study of the parasite and potential treatments.