5种鳎科鱼类核糖体ITS1序列比较

核糖体基因在很长一段时间内被认为严格遵循协同进化方式,但是在很多种类中都发现了明显的序列多态性,表明其是非协同进化。为了检测鳎科鱼类核糖体内转录间隔区1(ITS1)序列是否存在多态性,并探究其能否作为种类鉴定的分子标记,本研究克隆获得了5种鳎科鱼类共118条ITS1全序列。结果显示,眼斑豹鳎具有两种差异显著的片段类型,表明其在基因组中遵循非协同进化方式;而在其余4种鳎科鱼类中均没有发现序列多态性,表明其为协同进化。序列分析显示ITS1具有明显的种间长度异质性,最短的序列出现在蛾眉条鳎(412 bp),最长的为眼斑豹鳎(585 bp)。碱基分析显示ITS1序列在5种鳎科鱼类中都呈现出相同的趋势:CGAT,且GC含量为69.5%,远高于AT含量。聚类分析显示除眼斑豹鳎外,所有鳎类均单独聚为一支,种类区分度非常明显,表明ITS1序列能够作为种类鉴定的分子标记。但是眼斑豹鳎的一个克隆和东方箬鳎聚为一支,这种序列多态性对种类鉴定产生了干扰,因此用具有多态的ITS1序列作为分子标记时一定要有足够的克隆数量,避免因数据不充分而得到不正确的结论。     

珠母贝属6个种的ITS 2分子标记研究

对珠母贝属的大珠母贝、珠母贝、白珠母贝、黑珠母贝、长耳珠母贝、黑珠母贝和合浦珠母贝6个种的内部转录间隔区2(ITS2)序列及其两侧的5.8S和28S的部分序列进行了比较分析。其中黑珠母贝的序列来自GenBank。PCR扩增片段大小为600bp左右,测序结果表明,ITS2长211～254bp,两端的5.8S和28S分别长84bp和272bp(均含引物)。序列比对分析结果表明,5.8S和28S序列高度保守,不适合于种类鉴定,而ITS2序列高度变异,270个比对位点中有146个位点发生突变,其中72个位点发生插入/缺失突变。除白珠母贝和黑珠母贝之间的遗传距离较小外,其余种类之间的遗传距离远远大于种内遗传距离。基因型分析表明,每个种具有各自特有的基因型。基因型和序列变异分析表明ITS2序列可作为珍珠贝种类鉴定的分子标记。可用于种间、杂交育种、幼体和珍珠贝肉等材料的种类鉴定与遗传分析。     

快速鉴定四大家鱼早期资源种类的PCR方法

根据线粒体控制区序列设计了青鱼(Mylopharyngodon piceus)、草鱼(Ctenopharyngodon idellus)、鲢(Hypophthalmichthys molitrix)、鳙(Aristichthys nobilis)物种特异的PCR引物,通过对四大家鱼受精卵的PCR扩增,种间交叉扩增,相关种类早期资源的扩增以及未知种类鱼卵的鉴定发现,所设计的引物具有很高的种类特异性,对特定种DNA具有良好的扩增效果,但是不能进行交叉扩增,对四大家鱼外的其它种类也不能扩增。实验没有出现一例假阳性或假阴性结果,说明所设计引物的种类特异性高。该方法特别适合从鱼类早期资源大量样本中准确快速鉴定四大家鱼种类。     

4 种大型水母类ITS-5.8S rDNA 序列分析及其在钵水母类系统分析中的应用

由于钵水母类生物地理学研究的缺乏以及不同时期形态变异较大等原因,对其分类鉴定比较混乱和困难。为弥补形态学分类的缺陷,采用通用引物PCR扩增法,测定了分布于黄海北部和辽东湾海域海蜇(Rhopilema esculentum)、沙蜇(Nemopilema nomurai)、海月水母(Aurelia sp.)、白色霞水母(Cyanea nozakii)4种大型水母的ITS-5.8S rDNA序列,同时利用Gen Bank数据库中已有的钵水母纲(Scyphomedusae)ITS1(the Ribosomal First Internal Transcribed Spacer)同源序列对其进行序列分析并构建系统树,分析ITS1序列片段在大型水母种类鉴定方面的可行性及其在钵水母类系统及演化中的应用。结果显示,4种水母的ITS-5.8S rDNA序列变异较大且具有明显的序列长度多态性,序列长度范围675~833 bp。钵水母纲很多种类的ITS1序列具有种内长度多态性现象,这种长度多态性主要是由于微卫星重复次数不同所造成的。钵水母纲科间遗传距离为0.295~0.491,种间遗传距离为0.024~0.812;除白色霞水母和海蜇外,种内个体间遗传距离为0.000~0.099。采用ML法(maximum likelihood)和贝叶斯法(Bayesian)构建的分子系统树拓扑结构不完全相同且与形态分类学的观点不太一致。研究表明,ITS基因序列在钵水母纲不同阶元间变异较大,适合于钵水母纲种类鉴定和属内种间水平的系统进化研究。     

基于DNA条形码对温州洞头海域游泳生物的鉴定

温州洞头海域是浙南重要的渔场,具有丰富的渔业资源。为了监测该地区的游泳生物资源,在传统形态学鉴定的基础上,使用线粒体细胞色素C氧化酶亚基I基因(COI)对渔获物样品进行了鉴定分析。结果显示,所获得的生物隶属于4个纲19个科25个属的28个种,DNA条形码的有效鉴定率为96.5%。系统进化树分析显示,所有序列与GenBank数据库下载的同一种类序列都聚为一支,该结果可以有效证实鉴定结果的有效性。研究结果表明,将传统形态学鉴定与DNA条形码识别结合可以有效鉴定物种,有助于对洞头海域游泳生物的种类进行识别,并为这些游泳生物建立可靠的DNA条码参考库,为该地区渔业监测、海洋生物资源保护、海洋保护区的建立提供可参考的理论基础。     

基于COI和16S rRNA基因片段鉴定厦门海域的仔稚鱼

本文基于线粒体细胞色素C氧化酶亚基I(COI)基因和16S rRNA基因片段对采集于厦门海域的仔稚鱼样品进行种类鉴定,探究其在仔稚鱼种类鉴定中的适用性。研究共获得64条COI基因序列和74条16S rRNA基因序列,通过序列比对,COI基因将仔稚鱼样品鉴定为26个种类,其中19个种类鉴定到种、6个鉴定到属、1个种类仅鉴定到科;16S rRNA基因将仔稚鱼样品鉴定为29个种类,其中23个种类鉴定到种、6个鉴定到属。COI基因的平均种内遗传距离为0.001 5,平均种间遗传距离为0.197 6,16S rRNA基因的平均种内遗传距离为0.000 3,平均种间遗传距离为0.089 2,COI和16S rRNA基因的平均种间遗传距离都为平均种内遗传距离的10倍以上,两者都可以进行有效的仔稚鱼种类鉴定。在基于COI和16S rRNA基因构建的系统进化树上,所有物种都分别单独聚为一支,同一个种类的不同个体都能聚在同一个分支,这些物种均能得到有效区分。以上结果表明,COI和16S rRNA基因均可以实现仔稚鱼的种类鉴定,两种基因结合使用可以提高仔稚鱼种类鉴定的准确性。     

DNA条形码在厦门湾鱼卵和仔稚鱼分类鉴定中的应用

本研究选取2017年4月采集的厦门湾鱼卵、仔稚鱼样品进行DNA条形码分析。获得鱼卵、仔稚鱼的COI基因序列27条,共鉴定种类11种,全部鉴定到种,隶属于4目9科11属。在系统进化树的分析中,同一种类的不同个体都聚在一支,所有物种都分别聚为独立的一支,这些物种都能有效地区分开来,说明利用COI基因可以进行有效的仔稚鱼物种鉴定。同时获取11种鱼类浮游生物高清图像,并描述其甄别性的形态特征。以上结果表明,线粒体COI基因作为DNA条形码可以实现鱼卵和仔稚鱼的物种鉴定,且较多能鉴定到种的水平。     

凡纳滨对虾内源优势腐败菌的分离和鉴定

为了确定凡纳滨对虾在冷藏条件下内源优势腐败菌的种类,实验采用了细菌分离纯化法,将分离得到的内源腐败菌菌株进行分解蛋白质对比实验,筛选出分解蛋白质能力较强的菌株,进一步采用细菌自动生化系统鉴定法以及16S rRNA序列测定法进行鉴定,实验结果显示凡纳滨对虾在冷藏条件下的内源优势腐败菌是短杆菌属细菌,可以通过控制短杆菌属细菌的方法进行研究对虾内源保鲜技术。细菌自动生化系统鉴定结果的相似度为98.8%,16S rRNA序列鉴定结果的相似度为99.065%。     

基于DNA条形码技术的永暑礁泻湖鱼卵鉴定研究

为了解南海珊瑚礁鱼类的产卵时间、产卵地点和种类分布,本研究以南海永暑礁泻湖鱼卵为研究对象,根据形态学特征和DNA条形码序列信息对其进行种属鉴定分析。71个鱼卵样品根据形态学特征分为圆形和椭圆形2种类型,圆形鱼卵44个,椭圆形鱼卵27个。利用DNA条形码技术扩增测序获得了30个鱼卵样品的线粒体COI条形码序列,经BOLD数据库比对分析显示30个鱼卵样品均可鉴定到种,分属于1个目,7个科,13个种。双斑栉齿刺尾鱼有10个鱼卵,白尾栉齿刺尾鱼有4个鱼卵,为该海域的优势种类,其余各种类均为1~2个鱼卵。根据线粒体COI序列计算种内的K2P(Kimura-2-parameter)遗传距离为0.002,同一个属种间的遗传距离为0.004~0.132,而同一个科不同属间的遗传距离为0.117~0.185。该研究结果表明DNA条形码技术可以作为鉴定南沙群岛珊瑚礁海域鱼卵的有效方法,能被广泛应用于南沙群岛珊瑚海区域鱼卵和仔稚鱼的鉴定工作。     

基于DNA条形码技术的轮叶黑藻害虫——小筒水螟鉴定研究

根据DNA条形码序列信息和形态特征,对某严重危害虾、蟹塘轮叶黑藻的"卷叶虫"种类进行了种属鉴定分析。COⅠ基因序列信息显示,该"卷叶虫"幼虫和飞蛾的序列一致性为100%,为同一物种,飞蛾为"卷叶虫"幼虫的成虫。经Gen Bank数据库BLAST鉴定,为Parapoynx diminutalis,中文名为小筒水螟。幼虫全身长满刚毛、具卷叶行为,飞蛾呈浅白色、长有棕色斑块,上述特征与小筒水螟一致。综合上述依据,最终鉴定该"卷叶虫"为小筒水螟。     

Imported leishmaniasis of dogs: clinical aspects, diagnosis and therapy with Na stibogluconate (Pentostam)

Between May 1982 and September 1986 13 dogs with leishmaniosis received inpatient treatment with Pentostam at the I. Medical Department of the Veterinary Faculty, University of Munich. Predominant clinical signs of all animals were alterations of the skin and hair coat and additionally they showed a reduced general condition (4 dogs), loss of weight (3 dogs), diarrhea (5 dogs) and pain during movements. In the blood samples taken prior treatment in 9 dogs there was an increased total protein concentration. 5 dogs had changes in the red and 4 dogs in the white blood count. Thrombocytopenia occurred in 6 dogs. Leishmaniosis was diagnosed clinically and serologically (IFAT). Na-Stibogluconate (Pentostam) was used for the treatment at a dosage of 10 or 20 mg/kg on 10 or 14 consecutive days. Two dogs died during an early stage of therapy. 6 dogs were discharged as clinically cured after a single or repeated treatment course(s).     

嗜水气单胞菌灭活疫苗免疫后大鲵外周血免疫指标的变化

以福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)作为免疫原(F-AH), 通过腹腔注射免疫健康大鲵(Andrias davidianus), 分别于免疫后的第1、4、7、14、21、28天采集血液样品, 进行大鲵外周血液的血细胞计数与白细胞组成分析, 测定吞噬细胞的吞噬活性以及血清中和抗体效价, 于免疫28 d后进行攻毒感染试验。结果表明, 与对照组相比, 在免疫后第4、7、14和第21天, 免疫大鲵外周血中血细胞数量显著增加, 其中, 红细胞和白细胞数量均于第4天达峰值, 分别为7.83×10⁷个/mL和6.74×10⁶个/mL; 嗜中性粒细胞百分比也在第4天达峰值, 为28.60%, 单核细胞百分比在第7天达峰值, 为10.53%; 吞噬细胞活性显著提高, 且吞噬百分比和吞噬指数均在第4天达到最高值, 分别为34.09%和3.73。随后, 淋巴细胞百分比和中和抗体效价显著增加, 均在第21天达峰值, 分别为75.30%和1︰426.67。攻毒感染实验结果表明, 免疫组的相对免疫保护率为69.23%。由此可见, F-AH免疫原能够通过促进血细胞数量的增加、吞噬细胞吞噬活性增强以及特异性抗体的产生等方式提高大鲵的免疫保护力。

     

Use of the Reflotron system for laboratory diagnosis in swine and sheep

The "Reflotron" system (Boehringer Mannheim) was compared with standard laboratory methods for determination of blood values, e.g. haemoglobin, glucose, urea, ASAT/GOT and GGT in pigs and sheep. Various different commercial control sera as well as blood and plasma samples from pigs and sheep were used for evaluation of its precision. The day-to-day precision of all parameters was satisfactory. The accuracy of the values was tested in 56(48) blood samples of healthy and sick pigs (sheep) by using standard substances (glucose, urea) and by comparing them with values measured by standard laboratory methods. There were systematic differences between "Reflotron" and other values in the glucose- and enzyme-values, which can be corrected with the help of conversion factors. In pigs, the enzyme GGT can not be determined accurately with the "Reflotron" system.     

鲫血髓过氧化物酶的表达及其与血药浓度的关联性

为了研究在吡喹酮代谢过程中,鲫血髓过氧化物酶(myeloperoxidase,MPO)m RNA表达与血药浓度的相关性,以体重为(80±10.5)g的鲫鱼(Carassius auratus)为试验动物,单剂量(10 mg/kg)口灌吡喹酮(praziquantel,PZQ)后,在鲫血液中选取β-actin为内参基因,通过设计特异性引物,利用荧光定量PCR,分析了不同时间点鲫血MPO m RNA水平的相对表达量的变化;利用高效液相色谱法(high performance liquid chromatography,HPLC)测定了吡喹酮在鲫鱼体内的血药浓度,分析两者之间的相关性。结果显示,吡喹酮能够迅速进入血液,并被快速消除,药时数据符合二室开放模型。在1 h时,血液中吡喹酮的浓度达到最大值,为2.85?g/m L,96 h后血液中检测不到吡喹酮;灌药后,MPO基因表达量随时间呈先升后降趋势,在1 h时髓过氧化物酶表达量最高。此外,0.25 h、0.5 h、1 h、3 h与6 h组与对照组差异性极显著(P0.01),12 h组与对照组差异显著(P0.05)。48 h与96 h组与对照组差异不显著(P0.05)。相关性分析发现,MPO m RNA相对表达量与血液中吡喹酮的浓度之间相关系数r=0.96,为高度相关,并且推测MPO可能参与吡喹酮的氧化代谢。结论认为:(1)MPO m RNA相对表达量的升高与外源性药物吡喹酮的摄入有关。(2)血液中吡喹酮的残留量与MPO m RNA相对表达量线性相关。本研究旨在提供一种从分子水平评价水产动物体内药物残留的新思路。     

Blood and liver biopsy for the non‐destructive screening of tilapia lake virus

Detection of tilapia lake virus (TiLV) in tilapines is mainly from visceral organs of killed fish. However, lethal sampling might not be viable to broodstock and economically important ornamental cichlids. To contribute towards screening of the virus in asymptomatic infected fish, a subclinically infected population of Nile tilapia adults obtained from a local farm was preliminarily tested to compare different non‐lethal sampling methods, for example liver biopsy, gill biopsy, fin clip, mucus, faeces and blood for detection of TiLV. Only liver and blood samples gave positive results by PCR. Since blood sampling is relatively simpler, it was further used for five naturally co‐cultured juvenile fish species from above‐mentioned farm including 40 red tilapia broodstock and 20 Nile tilapia adults from two other different farms. The results showed that from the tested fish, 4 of 5 Nile tilapia, 2 of 5 hybrid red tilapia and 3 of 5 giant gourami blood samples tested positive, while 38 of 40 blood samples of red tilapia tested positive for TiLV in second‐step PCR. Sequencing representative PCR amplicons of positive samples confirmed sequence identity to TiLV. In conclusion, both blood and liver biopsy are practical non‐destructive sampling platforms for TiLV screening in cichlids with blood being more convenient, especially for tilapia broodstock.     

Clinical and bacteriologic studies of the frequency of mastitis during and after parturition in heifers lactating for the first time

The udders of 100 heifers were examined for clinical changes during parturition. Of each quarter colostral samples were taken and analyzed bacteriologically. 35 heifers (13.5% of the quarters) showed clinical changes of the quarters and/or of colostral samples. 14 of these animals (4.75% of the quarters) suffered from cellulitis-like mastitis. 13 (5.25% of the quarters) had acute catarrhalic mastitis and eight heifers (3.5% of the quarters) showed acute galactophoritis. In all milk samples of animals with pathological changes of the udder or of colostrum the causing bacteria could be identified. Besides the group of 35 heifers with clinical lesions udder-pathogenic bacteria were found in samples of 24 apparently healthy animals (20.5% of the quarters) during parturition. However, these animals failed to show clinical signs of inflammation at least until the end of this study (10 days post partum). Isolated bacteria included facultative and obligate pathogenic species, 70% of which were gram-positive cocci (i.e. Streptococcus [Sc.] uberis, Sc. dysgalactiae, Sc. agalactiae, Staphylococcus aureus and other staphylococci) versus 30% of gram-negative bacteria (i.e. Escherichia [E.] coli, Klebsiella and other coliform species. In-vitro-susceptibility tests showed best effectiveness of Penicillin against Sc., Cloxacillin against staphylococci and Gentamycin and Polymyxin B against coliform bacteria. Possible pathways, leading to bacterial infection of first lactating heifers during parturition, are discussed. Pathways of infection seem to be of lesser importance for the development of apparent, sometimes severe mastitis with coliform bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmission of lymphocystis disease virus to cultured gilthead seabream,Sparus aurata L., larvae

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR‐hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF‐1 cells. Whole‐mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV‐positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR‐hybridization) or in larvae (PCR‐hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR‐hybridization, ISH and IHC. After feeding on LCDV‐positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.     

珠江河口重金属镉的含量与分布的季节特征

对珠江河口水体、沉积物和生物体中重金属镉(Cd)的含量与分布进行调查研究,结果表明,水体中Cd含量范围在0.022～0.047 1 mg/L,并且Cd含量在时间上由高到低依次为:2月、5月、11月、8月,2月Cd含量显著高于5月、8月和11月(P＜0.05).以海水水质标准衡量,珠江河口水体2008年中大部分时间为海水二类水质.沉积物中Cd含量范围在5.062～9.239 mg/kg,平均含量为6.974 me/kg,并且沉积物中Cd含量均超过海洋沉积物3类标准.与国内外河口比较,珠江河口沉积物中Cd含量处在较高水平,污染比较严重,并且有逐年升高的趋势.生物体中Cd含量范围在0.144～0.430 mg/kg,鱼类Cd含量均超过国家水产品卫生标准,并且肉食性鱼类Cd含量略高于杂食性鱼类,但差异不显著(P＞0.05).与往年调查进行比较发现,珠江河口鱼类Cd含量2000年较1988年有大幅增加,并且2000年以后增加也比较明显.     

The congenital portosystemic shunt in dogs and cats. I

An overview of the circulation of the liver and of the pathogenesis of hepatic encephalopathy as a result of portal vascular anomalies is given. Clinical signs associated with portal systemic shunts are described on the basis of 16 cases, 14 dogs and 2 cats. These animals ranged in age at the time of presentation from 4 months to 7 years. The predominant abnormality observed were central nervous signs, which differed in severity. 15 animals showed a reduction in liver size. The different techniques of contrast angiography allowing demonstration of a portal systemic shunt are presented along with a discussion of the pros and cons of each. Additionally the significance of making portal venous pressure measurements prior to each angiography is also explained. In most cases mesenteric portography was chosen. Based on their location the anomalies could be categorized as intrahepatic (4 dogs) or extrahepatic (10 dogs, 2 cats). In both groups breeds of various size are represented. The extrahepatic shunts could be further described as portal-caval (n = 5), portal-phrenic (n = 4) and portal-azygos (n = 3). In five of the older animals angiography showed in addition some hepatic perfusion by the portal vein. Laboratory evaluation revealed increased resting blood ammonia concentrations (greater than 200-912 micrograms/100 ml) in all animals. Seven dogs had definitely subnormal BUN concentrations (less than 10 mg%) and ten dogs low total plasmaprotein levels (less than 5.4 g%). Free amino acids (24) were determined in four dogs and a lowered hepatic encephalopathy index (less than 1.64) was found. Medical palliative therapy to control the clinical signs is discussed. The only effective long term therapy is, however, surgery. The shunt vessel is narrowed so that a greater volume of portal blood reaches the liver. Experience gained from the surgical therapy of 14 animals is presented. Ten of these survived well without requiring further therapy at a later time. Finally the etiology, prognosis, and differential diagnosis are summarized.