Detection of koi herpesvirus DNA in tissues of infected fish

A newly recognized herpesvirus, koi herpesvirus or KHV, causes a lethal disease in common carp, Cyprinus carpio , and its colourful strain known as koi or fancy carp. In this study, we report new outbreaks of the disease, present initial characterization of the KHV genome, and describe assays for detection of KHV DNA in infected cells and tissues of infected fish. Restriction endonuclease (RE) profiles of viral DNA derived from two epidemiologically distinct KHV isolates were identical to each other. Cloned KHV BamHI and SphI DNA probes specifically hybridized to KHV DNA, but not to DNAs derived from a variety of other fish herpesviruses. The KHV DNA probes detected KHV DNA in tissues of experimentally infected koi fish by DNA hybridization. The KHV specific polymerase chain assays (PCR) were developed for rapid detection and confirmation of KHV DNA in tissues of infected fish.     

Determination of quercetin concentrations in fish tissues after feeding quercetin-containing diets

Concentrations of quercetin in fish tissues were measured for the first time using HPLC-electrochemical detection method. Its identity was also ascertained with UV-photodiode array detection. Quercetin, in aglycone form, was at measurable concentrations in tilapia plasma, liver, and whole body homogenate when fed with diets containing 1% quercetin (aglycone) for 1 or 15 weeks. Hydrolysis with glucuronidase/sulfatase treatment for the purpose of cleaving conjugates did not increase quercetin levels, suggesting that glucoronide or sulfate conjugates are not the major metabolic forms in Nile tilapia (Oreochromis niloticus). No quercetin was detected in plasma of rainbow trout (Salmo gairdneri) or white sturgeon (Acipenser transmontanus) fed commercial diets. The results suggest that quercetin is absorbed in tilapia and that this flavonoid is deposited mainly in aglycone form in the body after absorption.     

Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues

The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin‐fixed, paraffin‐embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)‐based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat‐shock protein (hsp65) gene. PCR‐restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false‐negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (～30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR‐based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax‐embedded and formalin‐fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies.     

鳜黏膜淋巴组织结构的初步研究

采用组织学和电镜技术,应用HE常规染色方法、AB-PAS黏液细胞染色方法、免疫组织化学方法,对鳜(Siniperca chuats)黏膜淋巴组织的基本结构以及免疫相关细胞的形态、类型及其在黏膜淋巴组织中的分布情况进行了研究.结果表明,鳜的鳃弓、鳃小片基部、皮肤的表皮以及肠上皮中存在大量的黏液细胞,用AB-PAS染色方法将鳜黏液细胞分为6种类型;免疫组化方法可在鳃的血窦腔、皮肤的基底层以及肠黏膜层中检测到抗体分泌细胞的存在;透射电镜观察显示,黏膜淋巴组织中存在着黏液细胞、淋巴细胞、浆细胞、吞噬细胞、肥大细胞、朗罕氏细胞、嗜中性粒细胞等免疫相关细胞.结论认为,鳜黏膜淋巴组织具有完成免疫应答的细胞基础.     

Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non‐salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold‐conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13‐nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody‐coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 10⁴ CFU mL^?1, and results were obtained within 45 min. The antibody‐coated gold nanoparticles were stable for at least 2 months at 4°C. The immunoassay using antibody‐coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.     

贵州百花湖鱼体器官及肌肉组织中重金属的分布特征及其与水体重金属污染水平的相关性

通过对鳃、肝脏、肾脏、心脏、背肌、腹肌和尾肌等器官与肌肉中6种重金属(Pb、Cd、Cu、Cr、As和Hg)的调查, 分析了百花湖野生鲢和鲤体内重金属含量水平、分布特征及其与水体重金属之间的相关性。在所测4种器官和3处肌肉组织中, 鲢和鲤体内重金属平均含量由高到低的顺序为 Cu > Pb > Cr > Cd > As > Hg; 在鲢和鲤各器官中, Cr和Cu表现出较强的相关性, 其相关系数分别为r=0.878(P<0.01)和 r=0.972(P<0.01), 而Cr和Hg只在鲤体内表现出负的相关性 r= -0.782(P<0.05); 以6 种重金属为依托的鱼体器官自身之间的相关性较强, 但各器官与沉积物和水质中重金属平均含量之间的相关性较弱。聚类分析表明, 在两种鱼体内, 这6 种重金属可以分为3类。研究结果表明, Pb主要蓄积在鲤的肝脏和心脏中, Cd主要存在于两种鱼的肾脏中, Cu 主要在肝脏及心脏中蓄积, Cr主要蓄积在两种鱼体的肝脏和心脏中, As在鲤心脏中含量较为突出, 但在鲢各器官中含量差异不大, Hg在整个鱼体器官及组织中的含量较为均匀, 鱼体可食用的肌肉组织中这6种重金属含量普遍较低。沉积物中重金属主要以硫化物形态存在, 生物可利用度低, 可能是鱼体重金属元素平均含量普遍较低的主要原因。     

Distribution and properties of lactate dehydrogenase isoenzymes in red and white muscle of freshwater fish

The distribution and kinetics of LDH isoenzymes in red and white muscles of 5 species of salmonids, 4 species of cyprinids and one coregonid species were studied. In all species the white muscles are characterized by the occurrence of only the most cathodic isoenzymes, or groups of isoenzymes. The red muscles contained either the full set of isoenzymes (cyprinids) or a selection in which the anodic forms dominated (salmonids, coregonid). The most striking difference between the two types of muscle was met inCoregonus sp. The temperature profiles of pyruvate affinity are similar in all species of fish studied. On the other hand, K_m(pyr) values and degree of pyruvate inhibition are closely related and vary greatly with temperature, with the taxonomic position (and thus biology) of the species, and with electrophoresic mobility of the isoenzyme. Highest affinity and strongest inhibition occurred in the anodic (H₄) isoenzymes of cyprinids at low temperature; lowest affinity and zero inhibition in the cathodic isoenzymes (M₄ M₄) of salmonids and coregonids at high temperature. In salmonids the more recently duplicated loci of the M-group of isoenzymes possess identical K_m values at all temperatures, whereas the two older M and H loci differ greatly in this respect. Thus the more recent duplication of LDH loci in salmonids and coregonids may be seen as a mechanism by which the tetramers required for LDH activity can be constructed from more closely related subunits than are provided by the older M and H loci.Some problems in connection with the determination of the kinetic constants of the lactate oxidase reaction are discussed and it is suggested that an alkaline, pyruvate trapping system provides conditions which are more realistic than those of other assay systems. The K_m(lactate) values found are in the biological range and, at 20°C, provide further circumstantial evidence that the red muscles of fish should be capable of oxidizing the lactate produced by the white muscles during strenuous exercise. At 4°C the K_m(lactate) values are abnormally high in all muscle preparations and thus are not correlated with the K_m(pyruvate) values which are lowest at this temperature.     

水环境Cu2+对鲫鱼组织Na+-K+-ATPase酶活力的影响

研究水环境铜对鲫鱼组织Na -K -ATPase酶活力的影响,探求Na -K -ATPase酶作为铜暴露生物标记物的可能性。以30尾鲫鱼作为受试生物,随机分成5组,每组6尾。研究其剂量效应,铜离子浓度分别为0、0.004、0.02、0.1、0.5 mg/L。结果表明,随着Cu2 浓度的增加,各组织(肝、肾、鳃)的Na -K -ATPase酶活力均呈现出先升高、后降低的现象;随着Cu2 浓度的进一步上升,实验组酶活力显著低于对照组(P<0.05)。Cu2 对鳃组织Na -K -ATPase酶活力的影响最大,其次为肝,对肾的影响较小。Cu2 的胁迫可引起鲫鱼组织酶活力发生变化,Na -K -ATPase酶有望成为一个较好的水环境污染早期预警指标。     

Retention of formaldehyde in the tissues of two tropical fish species following exposure to therapeutic levels

Abstract. Formalin is an effective fish chemotherapeutant which is used to control certain ectoparasitic and bacterial infections. It is toxic to fish and its pathological and physiological effects have been reported. An experiment was conducted to elucidate the possibility of retention of formaldehyde in the muscles of two tropical food fish species, following exposure to therapeutic levels of formalin. The results indicated that Puntius gonionotus (Bleeker), retained 5·75 ppm formaldehyde following exposure to 50 ppm formalin for 24h. In contrast, Clarias batrachus (L.) retained 14·03 ppm and 18·72 ppm formaldehyde following exposure to formalin at 50 ppm and 100 ppm for 24 h respectively. However, no formaldehyde was detected after keeping the exposed fish in clean running water for 24h.     

Lactate dehydrogenase isozymes in the trunk and cardiac muscles of an antarctic teleost fish,Notothenia neglecta Nybelin

The distribution and kinetics of lactate dehydrogenase (LDH) isozymes in the red and white trunk muscles, and cardiac muscle of an antarctic teleost fish (Notothenia neglecta Nybelin) have been studied. Pyruvate inhibition of LDH in all three muscle types is very low, being less than 50% even at a concentration of 60mM pyruvate. Activity versus pyruvate concentration profiles are not significantly different for LDH in all three muscle types. The Michaelis constant (Km) for pyruvate was not significantly different for all three LDH's. Raising the assay temperature caused an increase in Km of similar form in all three muscle types, while Km was lowest at the lowest assay temperature (–1°C). When samples were run on a polyacrylamide gel, the bands stained specifically for LDH activity appeared at identical positions as those of the H2M2 band of the standards.It would appear therefore that the LDH isozyme found in the red and white trunk muscle ofN. neglecta is identical to that in cardiac muscle. This fact is discussed in relation to the physiological ecology of antaretic fishes, and the metabolic constraints imposed by their habitat, including their apparent low capacity for utilising glycolytic fuels.     

牙鲆抗菌肽hepcidin基因在成鱼组织和不同发育时期胚胎中的表达分析

抗菌肽hepcidin是由hepcidin基因编码产生的小分子多肽,是机体天然免疫的重要因子。本文利用RT-PCR的方法分析表明,抗菌肽hepcidin基因在牙鲆成鱼不同组织和不同发育时期胚胎中普遍表达,但是表达水平存在着明显的差异。     

Response of ATPases in the osmoregulatory tissues of freshwater fish Oreochromis niloticus exposed to copper in increased salinity

An increase in salinity of freshwater can affect the physiology and metal uptake in fish. In the present study, Nile tilapia Oreochromis niloticus were exposed to copper (1.0 mg/l) in increased salinities (2, 4, and 8 ppt) for 0, 1, 3, 7, and 14 days. Following the exposures, the activities of Na⁺/K⁺-ATPase, Mg²⁺-ATPase, and Ca²⁺-ATPase were measured in the gill, kidney, and intestine to evaluate the changes in osmoregulation of fish. Results showed that increases in salinity and Cu exposure of fish significantly altered the ATPase activities depending on the tissue type, salinity increase, and exposure durations. Salinity-alone exposures increased Na⁺/K⁺-ATPase activity and decreased Ca²⁺-ATPase activity. Na⁺/K⁺-ATPase activity decreased following Cu exposure in 2 and 4 ppt salinities, though the activity increased in 8 ppt salinity. Ca²⁺-ATPase activity decreased in the gill and intestine in all salinities, while the activity mostly increased in the kidney. However, there were great variations in Mg²⁺-ATPase activity following exposure to salinity alone and salinity+Cu combination. Cu accumulated in the gill and intestine following 14 days exposure and accumulation was negatively correlated with salinity increase. Data indicated that ATPases were highly sensitive to increases in salinity and Cu and might be a useful biomarker in ecotoxicological studies. However, data from salinity increased freshwaters should carefully be handled to see a clear picture on the effects of metals, as salinity affects both metal speciation and fish osmoregulation.     

冬夏两季五种经济鱼类组织脂肪酸含量及组成分析

为了解不同季节不同鱼类不同组织中的不同脂肪酸含量,科学地指导鱼类膳食消费,本实验研究了冬夏两季,采集自上海市场常见的5种经济鱼类:大黄鱼(海洋肉食性),银鲳(海洋杂食性),日本鳗鲡(淡水肉食性),莫桑比克罗非鱼(淡水杂食性),草鱼(淡水草食性),分别检测鱼背部肌肉、腹部肌肉、尾部肌肉、肝脏和腹腔脂肪组织的脂肪含量和脂肪酸绝对含量。结果显示,5种鱼肌肉脂肪酸谱存在显著差异,并与各自的生活环境及食性均有关系;在鱼的腹腔脂肪或肝脏中,饱和脂肪酸(SFAs)和单不饱和脂肪酸(MUFAs)含量较高,且与组织脂肪含量密切相关;而多不饱和脂肪酸(PUFAs)、n-3系多不饱和脂肪酸(n-3 PUFAs)和n-6系多不饱和脂肪酸(n-6 PUFAs)含量较低,且与组织脂肪含量关系不大;大黄鱼和银鲳各肌肉组织中的n-3 PUFAs、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)含量以及n-3/n-6值高于莫桑比克罗非鱼和草鱼,并与组织脂肪含量呈正相关;冬季草鱼腹部肌肉、莫桑比克罗非鱼尾部肌肉以及日本鳗鲡和银鲳的肝脏中的n-3 PUFAs含量较夏季高。研究表明,脂肪酸组成与物种、食性、水域环境以及季节温度和组织部位均有关系。从补充n-3 PUFAs摄入的角度分析,日本鳗鲡、大黄鱼和银鲳营养价值高于莫桑比克罗非鱼和草鱼,冬季鱼类的营养价值高于夏季。     

斑鳜和翘嘴鳜不同组织中过氧化物酶、酯酶和乳酸脱氢酶的比较研究

采用聚丙烯酰胺垂直平板凝胶电泳技术,分别取斑鳜的肌肉、肝脏、眼、心脏、脑、肾脏6种组织和翘嘴鳜的肌肉、眼、脑、肾脏4种组织进行过氧化物酶(POD)、酯酶(EST)、乳酸脱氢酶(LDH)分析。结果表明:LDH同工酶的组织特异性不高;EST同工酶在斑鳜、翘嘴鳜的对应组织中(肾、眼、肌、脑)都存在不同程度的差异;POD同工酶具有种间特异性,可以作为区分这两种鳜鱼的依据。     

Evaluation of the AQUARAPID-Va, AQUAEIA-Va and dot-blot assays for the detection of Vibrio anguillarum in fish tissues

The comparative accuracy of the serological assays AQUARAPID-Va, AQUAEIA-Va (BIONOR AS), and dot-blot for a rapid diagnosis of vibriosis in fish was evaluated. Twenty-one Vibrio anguillarum strains, representative of pathogenic and environmental serotypes, and 13 strains of other fish pathogenic bacteria were used to assess the sensitivity and specificity of the detection methods. The serological assays tested detected all the strains of V. anguillarum serotypes O1 and O2. The dot-blot assay was the most specific and sensitive method, detecting almost all isolates from serotypes O1, O2 and O3, with an average sensitivity of 1 x 10(6) bacteria g(-1) of fish tissue. The AQUARAPID-Va and the AQUAEIA-Va systems were able to detect 5 x 10(6) and 5 x 10(7) bacteria g(-1) of fish tissue, respectively. The simplicity, effectiveness and speed of the AQUARAPID-Va system confirmed this method as the most suitable serological test for the detection of V. anguillarum in field analysis and small-scale laboratory studies.     

黄颡鱼肝脏胞质中NADP依赖性的异柠檬酸脱氢酶(IDPc)的分离纯化和酶学性质

采用Sephadex G-100凝胶过滤、DEAE Sepharose离子交换、Sephadex G-25凝胶过滤等方法对黄颡鱼肝脏胞质中NADP依赖性的异柠檬酸脱氢酶(IDPc)进行纯化,并研究其相关的酶学性质。结果显示,IDPc的比活力为7.94 U/mg,亚基分子量为36.7 ku。IDPc活性最大时的pH和温度分别为8.0和65℃,活化能为81.33 kJ/mol,底物米氏常数KmNADP和KmIC分别为0.056和0.175 mmol/L,底物最大反应速率VmNADP和VmIC分别为9.04和10.51U/mg,催化效率KcatNADP和KcatIC分别为0.16和0.06 min/mg,产物NADPH对IDPc表现为竞争性抑制,抑制常数KiNADPH为0.034 mmol/L。IDPc的催化作用强烈依赖于金属离子Mn2+、Mg2+,没有金属离子存在时反应几乎不进行,二价金属离子激活IDPc的顺序为Mn2+Mg2+Zn2+Ca2+Cu2+,Cu2+几乎不能激活IDPc的活性。Ca2+和Zn2+既是激活剂也是抑制剂,与其作用浓度有关,Cu2+基本上对其无影响。通过对IDPc系统的酶学性质探讨,能够为深入研究IDPc催化与调节机制奠定基础。     

Screening for organic acids in fish tissues with special reference to the distribution of taurine inRutilus rutilus L.

A liquid chromatographic method for rapid profiling of some organic acids and other compounds of physiological interest in fish tissues is presented. The method provides the detection of -ketoglutaric, malic and pyruvic acid and is optimized for the quantitation of citric, phosphoric, succinic, lactic and fumaric acids, as well as glucose and especially taurine. These substances were separated in a single analytical run on a cation exchange column with dilute sulfuric acid as the elutant. The effluent was monitored by a refractive index or an UV detector at 208 nm wavelength. Resolution was satisfactory. No further treatment was found to be necessary when samples were deproteinized with perchloric acid. Taurine (2-aminoethansulfonic acid), not previously described in the roach (Rutilus rutilus L.), occurs in considerable amounts in red and white muscle and heart tissue. However, it is also present in all other tissues.