Histopathological studies of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis (Temminck & Schlegel)

A histopathological study was carried out on ayu, Plecoglossus altivelis, with bacterial haemorrhagic ascites. The fish were obtained from culture ponds in Wakayama Prefecture in 2003. The causative agent was identified as Pseudomonas plecoglossicida by a slide agglutination test using anti-P. plecoglossicida FPC941 serum. Histopathological studies revealed lesions in spleen, kidney, liver, intestine, heart and gills. Lesions in the spleen and haematopoietic tissue were prominent and invaded by P. plecoglossicida. Necrotic lesions accompanied by haemorrhage, fibrin deposition and oedema occurred in the splenic pulp and sheathed tissue, and in the kidney. The liver also had necrotic lesions and abscess formations. However, the intestine, heart and gills were only slightly invaded by P. plecoglossicida. No lesions or bacteria were observed in the brain.     

Detection and identification of Flavobacterium psychrophilum from gill washings and benthic diatoms by PCR-based sequencing analysis

A nested polymerase chain reaction (PCR) amplification technique was used to detect Flavobacterium psychrophilum from washings of fish gill surfaces and benthic diatoms as environmental samples. Gill washing samples were prepared from kawamutsu, Zacco temminckii (Temminck & Schlegel) and oikawa, Z. platypus (Temminck & Schlegel). Benthic diatom samples were collected from stone surfaces. All samples were collected from rivers in Wakayama Prefecture, Japan from November 2003 to January 2004. Following simple DNA extraction using a chelating resin, nested PCR techniques targeting 16S-rDNA and gyrB regions were performed, and PCR products were cloned and sequenced. With nested PCR amplification for the 16S-rDNA gene, ambiguous PCR products were detected from two of six samples, and by cloning and sequencing analysis were found not to be DNA fragments amplified from F. psychrophilum. Using nested PCR for the gyrB gene, however, five of six samples were clearly positive for F. psychrophilum in agarose gel electrophoresis, and were found to be identical with nucleotide sequences of F. psychrophilumgyrB deposited in DNA databases by sequencing analysis. Results indicate that nested PCR for the gyrB region is a useful technique to detect low levels of F. psychrophilum from environmental samples contaminated with many other organisms.     

A novel genotyping technique for distinguishing between Flavobacterium psychrophilum isolates virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel)

We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G‐C, A‐T, A‐C and G‐T). The G‐C type isolates exhibited strong pathogenicity to ayu, whereas the A‐T and G‐T types did not show any pathogenicity to this species. The A‐C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F. psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G‐C type) and those that are potentially not harmful or less harmful (A‐C, A‐T and G‐T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F. psychrophilum isolates.     

PCR-RFLP genotypes associated with quinolone resistance in isolates of Flavobacterium psychrophilum

A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.     

基于线粒体D-loop基因序列研究我国5种虾虎鱼类的系统进化关系

利用PCR技术扩增河川沙塘鳢(Odontobutis potamophila)、鸭绿沙塘鳢(Odontobutis yaluensis)、中华沙塘鳢(Odontobutis sinensis)、葛氏鲈塘鳢(Perccottus glenii)及尖头塘鳢(Eleotris oxycephala)的线粒体D-loop区并测序,获得河川沙塘鳢、鸭绿沙塘鳢、中华沙塘鳢、葛氏鲈塘鳢及尖头塘鳢线粒体D-loop区全序列分别为853bp、853bp、1028bp、852bp、848bp,结合GenBank中平头沙塘鳢(Odontobutis platycephala)、刺盖塘鳢(Eleotris acanthopoma)、黑体塘鳢(Eleotris melanosoma)、褐塘鳢(Eleotris fusca)、中华乌塘鳢(Bostrychussinensis)、云斑尖塘鳢(Oxyeleotris marmorata)、溪鳢(Rhyacichthys aspro))等7种虾虎鱼类同源的D-loop区序列,分析了这12种虾虎鱼类的系统发育关系。所使用的同源序列长度为832～846bp,其中保守位点452个,变异位点405个,简约信息位点258个,单态位点145个,转换/颠换平均值为0.8。基于Kimura2-parameter模型的12种虾虎鱼类遗传距离范围为0.043～0.312,在Kimura2-parameter模型构建的邻接树和非加权配对法系统树中,河川沙塘鳢、鸭绿沙塘鳢、中华沙塘鳢、平头沙塘鳢聚为一大支,刺盖塘鳢、黑体塘鳢、褐塘鳢、尖头塘鳢、溪鳢、葛氏鲈塘鳢聚为另一支。     

Species identification method for marine products of Seriola and related species

The complete nucleotide sequences of mitochondrial DNA (mtDNA) from four Seriola spp. (S. quinqueradiata, S. lalandi, S. dumerili, and S. rivoliana) were determined with the aim of developing a species identification analysis method for discriminating between commercially important Seriola spp. and other related species. In addition, the nucleotide sequences of the mitochondrial cytochrome b gene (Cytb) from five related but less expensive species in terms of market value (Seriolella brama, S. caerulea, S. punctata, Hyperoglyphe japonica, and Rachycentron canadum), which are often used as substitutes for Seriola spp., were determined. Restriction enzyme sites were examined by comparing the nucleotide sequences, and species-specific primers were designed for PCR-based restriction fragment length polymorphism (RFLP) analysis. Based on the results of the PCR amplification studies, the four Seriola spp. and the five related species tested could be categorized into three groups according to their PCR product pattern: a 373-bp product from the four Seriola spp., a 513-bp product from three Seriolella spp. and H. japonica, and a 204-bp product from R. canadum. In addition, RFLP analysis of the PCR products was able to differentiate these fish species.     

Growth of Flavobacterium psychrophilum in fish serum correlates with pathogenicity

Flavobacterium psychrophilum isolates, obtained from ayu, Plecoglossus altivelis, three species of salmonids and two species of cyprinids in Japan, were used in this study. Bacteria were inoculated to serum prepared from ayu or red spotted masu trout (RSMT), Oncorhynchus masou ishikawae, and incubated at 18 °C for 24 h. All isolates (n = 19) from ayu grew well with a 9- to 116-fold increase of CFU in ayu serum, while CFU decreased markedly in RSMT serum. In contrast, isolates (n = 17) from fish species other than ayu exhibited no growth in ayu serum, but some isolates from salmonids survived or grew (1.2-23.5 fold increase of CFU) in RSMT serum. The isolates that could not survive or grow in ayu and RSMT sera grew well in both heat-inactivated sera of ayu and RSMT. Experimental infection by intraperitoneal injection showed that ayu isolates examined were all pathogenic to ayu but not to RSMT, while none of the isolates from salmonids and cyprinids were pathogenic to ayu but some showed pathogenicity to RSMT. These results indicate that the in vitro growth ability of F. psychrophilum isolates in fish serum correlates well with their pathogenicity to fish, particularly in ayu.     

19株海水鱼致病性弧菌OmpK基因序列及其抗原性分析

从哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(V.alginolyticus)、副溶血弧菌(V.parahaemolyticus)克隆、测定了共19株海水鱼类致病性弧菌外膜蛋白OmpK基因序列,探讨其作为海水鱼类致病性弧菌共同抗原的分子基础.根据已知的弧菌外膜蛋白OmpK序列设计1对简并引物,利用聚合酶链式反应(PCR)方法从19株弧菌总DNA中分别扩增得到约800bp外膜蛋白OmpK的基因片段,将其克隆到pDM18-Tvector载体筛选阳性重组子进行序列测定.结果显示,OmpK基因分别含有786bp～849 bp的开放读码框,编码261～282个氨基酸,其核苷酸序列之间的相似性在72%～100%,推测氨基酸序列的相似性为71%～100%,且种内OmpK氨基酸序列的相似性比种间高.序列分析还表明,每一种弧菌OmpK基因都有一段特异性序列,可用于设计核酸探针或特异性引物来诊断、检测哈维氏弧菌等海水鱼致病性弧菌.本研究不仅从基因水平上证实了外膜蛋白OmpK广泛存在于海水鱼致病性弧菌中,而且证明了它们之间具有较高的相似性.由结果推测外膜蛋白OmpK是哈维氏弧菌、溶藻弧菌、副溶血弧菌等致病性弧菌的一种共同抗原,是较好的亚单位疫苗候选成分,为进一步研制广谱的海水鱼类致病性弧菌外膜蛋白基因工程亚单位疫苗提供了理论基础.     

TaqMan real-time polymerase chain reaction assay for rapid detection of Flavobacterium columnare

Flavobacterium columnare is a ubiquitous Gram-negative bacterium that causes columnaris disease in a wide variety of fish worldwide. Timely detection of this bacterium is important to prevent its spreading and to reduce the economic loss to fish farmers. We developed a TaqMan-based real-time polymerase chain reaction (PCR) targeting a 113 bp nucleotide region of the chondroitin AC lyase gene of F. columnare G₄. Specificity of the assay evaluated with 20 isolates of F. columnare and 15 other taxonomically or ecologically related bacteria revealed that the primers and probe were 100% specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures, over a range of dilutions [3.1 × 10⁰–3.1 × 10⁶ colony-forming units (CFU) mL⁻¹], was observed to be ∼3 bacterial cells. The lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear with the log of amount of nucleic acid (R²=0.994) over that range (5.4 ng–5.4 fg). In tissues (blood, gills and kidney) of F. columnare experimentally infected fish, the bacterial numbers measured by TaqMan real-time PCR ranged from 3.4 × 10⁰ to 9.5 × 10⁵ CFU mL⁻¹. In both F. columnare experimentally infected and spiked samples, positive PCR results were confirmed by bacteriological culture with 100% agreement. The TaqMan real-time PCR developed in this study is specific, sensitive and reproducible for the detection and quantitation of F. columnare in infected fish.     

Characterization of the DNA nucleotide sequences in the genome of red sea bream iridoviruses isolated in Korea

The nucleotide sequences of DNA fragments amplified by polymerase chain reaction (PCR) from four different genomic regions of nine red sea bream iridoviruses (RSIVs) isolated from different species of fish, different areas and in different years in Korea were compared with the reported reference sequences. One isolate, RSIV Namhae, showed 100% homology to the reference sequences, while the other eight isolates, which appeared to contain identical nucleotide sequences, showed 96.6–98.9% homology with reference sequences depending upon the target regions of PCR gene amplification. However, differences in nucleotide sequences were not apparent between the RSIVs isolated in different locations, in different years or in different host species. We also cloned and sequenced the 3′ end flanking region (K1) of the DNA polymerase (DPOL) gene using the cassette ligation-mediated PCR method. This sequence was 4436-bp long and possessed two open reading frames (ORF-1 and ORF-2) oriented in opposite directions. The putative proteins encoded by these two ORFs could not be characterized by comparison with the proteins of other species in the data banks. The presence of the ribonucleotide reductase small subunit (RNRS) gene at the 3′ end of the K1 region allowed us to determine that these two genes, RNRS and DPOL, are separated 5508 bp and oriented in the same direction in the genome of RSIV. Moreover, it is of interest that a PstI-restriction fragment, of which the sequence but not the location within the RSIV genome had previously been reported, is located at nucleotide positions from 1096 to 2054, extending from within the ORF-1 region, spanning the intervening sequence between ORF-1 and ORF-2, and extending into the ORF-2 region. Various repeating sequences up to 86 bp were present at the 3′ ends of ORFs, especially within the nucleotide sequences at the 3′ terminus of ORF-2. No similarities were detected when the DNA sequences of the K1 region were compared to the DNA sequences of a repetitive element in the genome of other iridoviruses.     

A sensitive FRET probe assay for the selective detection of Mycobacterium marinum in fish

Mycobacterium marinum is the causative agent of mycobacteriosis in wild and cultured fish and of atypical infection in humans. For the diagnosis of M. marinum , cultural and traditional polymerase chain reaction (PCR) methods are currently used. However, these protocols, although able to discriminate within Mycobacterium spp., have proved to be time-consuming or difficult to carry out. For this reason, the aim of this study was to obtain a rapid and specific diagnostic tool to quantify fish Mycobacterium spp. or to discriminate M. marinum from other mycobacteria. A primary PCR amplification with SYBR Green had a detection limit (dl) of 10² Mycobacterium DNA copies with a log-linear quantification range up to 10⁴ ( R ² = 0.99). The second PCR using FRET probes, flanking a region containing species specific nucleotide variations, was designed and validated with synthetic erp gene fragments corresponding to different mycobacterial species, different whole mycobacteria suspensions, experimentally infected fish tissues, tissues from experimentally infected fish, and samples of cultured fish. The results show that the FRET probes demonstrate a high specificity as the melting curve analysis allowed efficient discrimination of M. marinum from Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium pseudoshottsii , Mycobacterium shottsii and Mycobacterium ulcerans . The kidney is the organ with the strongest detection signal and using fish tissues the method has a mean sensitivity of 50 DNA copies/PCR.     

矛尾复鰕虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立

取体表出血、肌肉溃疡的虾蟹养殖塘混养大量死亡的矛尾复鰕虎鱼的深层溃烂组织及肝脏进行细菌分离,2种被检组织中均分离到2种优势生长菌落;人工感染试验表明,其中的一株分离菌(S090801)浓度为106～108 cfu/ml可引起矛尾复鰕虎鱼全部死亡,该菌的形态特征、生理生化特性及基于16SrRNA和gyrB 2种基因的系统发育学分析确定为弧菌属的哈氏弧菌。同时基于gyrB基因序列设计1对特异性引物,建立了一种哈氏弧菌快速、敏感的PCR检测方法,扩增目的片段大小为363bp,该方法检测哈氏弧菌模板DNA最低质量浓度为0.046875ng/μl,可用于哈氏弧菌引起的水产动物疾病的快速诊断及分子流行病学的调查研究。     

栉孔扇贝核糖体DNA转录间隔子序列研究及其潜在应用

以相应引物PCR扩增栉孔扇贝(Chlamys farreri)核基因组的核糖体DNA两个转录间隔子(ITS-1和ITS-2)，PCR产物经T载体连接后进行克隆、测序，分别得到了340bp和510bp的碱基序列，序列大小非常适合遗传变异及分子系统学研究。其A、T、G、C含量在ITS-1分别为32.06％，20.59％，22.35％和25.00％，在ITS-2分别为30.00％，21.37％24.12％和24.51％。这两个变异性较大的序列在扇贝种群中应用潜力很大，可广泛用于种内群体间遗传变异研究、种质鉴别及系统学研究。     

金焰笛鲷rDNA基因转录间隔区ITS-1序列分析

以持异性引物扩增了金焰笛鲷(Lutjamts fulviflamma)的核糖体第一转录间隔区(ITS-1),扩增产物经克隆后测序,测得 ITS-1长度为566 bp。其中 A、T、G、C 4种碱基的含量分别为14.1%、16.1%、30.2%、39.6%,G C(69.8%)含量明显高于 A T 含量(30.2%)。将此引物在笛鲷属其他4种鱼类中扩增,发现该对引物有很好的通用性;比较发现在不同种中 ITS-1存在着较大的差异,适合将其应用于分子系统学和种质资源方面的研究。     

Molecular typing by pulsed-field gel electrophoresis of Flavobacterium psychrophilum isolates derived from Japanese fish

Sixty-four isolates of Flavobacterium psychrophilum from ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel), and other fish (n=16) in Japan and the type strain (NCIMB 1947(T)) were typed using pulsed-field gel electrophoresis (PFGE) with endonuclease BlnI and XhoI. These isolates were classified into 20 clusters and 42 genotypes by PFGE analysis. The most predominant cluster of isolates from ayu was cluster XII (n=20), followed by clusters XVII, XVI, XX, XI, IX, X, XIII and XV; the remaining 17 isolates from other fish were divided into clusters I, II, III, IV, V, VI, VII, VIII, XIV, XVIII and XIX. The PFGE genotype of isolates from ayu clearly differed from those of other fish. The isolates from ayu in Gunma Prefecture belonged to clusters XII, XVI, XVII and XX, and the strains of three of these clusters (XII, XVII and XX) were isolated from ayu in 15 of 19 prefectures. PFGE typing enabled more accurate classification of isolates into clusters than previously achieved by analysing the restriction fragment length polymorphism of PCR products. These results suggest that F. psychrophilum isolated from ayu and other fish are genetically different and strains with several PFGE types have spread within Japan.     

A novel cytochrome P450 1D1 gene in Nile tilapia fish (Oreochromis niloticus): partial cDNA cloning and expression following benzo-a-pyrene exposure

To understand the detoxification and bioactivation mechanisms for organic contaminants, it is essential to identify the cytochrome P450 (CYP) complement. Therefore, this study aimed to clone a partial cDNA sequence of the novel CYP1D1 gene from the fish Oreochromis niloticus and examine whether intraperitoneal injection of benzo-a-pyrene (BaP), a potent AHR agonist, is capable of inducing CYP1D1 mRNA expression in different tilapia fish tissues. The cloned nucleotide sequence consisted of 713 bp representing a portion of the tilapia CYP1D1 cDNA ORF, encoding 237 amino acids. Amino acid sequence comparison of O. niloticus CYP1D1 with the sequences of CYP1D1 from other species showed that this gene shared the highest identity of 81% with Fundulus heteroclitus CYP1D1. Furthermore, analysis of the percent identities shared by the deduced amino acid sequence of O. niloticus CYP1D1 with the sequences of CYP1 from other species revealed that the highest identities were shared with fish CYP1As. Real-time PCR results revealed that the highest expression level of CYP1D1 mRNA was found in muscles, followed by gills, liver, and intestine, while there was no detectable expression recorded in bile acid. These results indicate that tilapia CYP1D1 plays an important role in the metabolism of xenobiotics, expanding our knowledge regarding the diversity of CYP1 genes in this important model fish species.

     

暗纹东方鲀线粒体DNA控制区结构和系统发育分析

以暗纹东方(Takifugu fasciatus)肝脏线粒体DNA为模板,参照GenBank中红鳍东方(Takifugu rubripes)线粒体DNA序列设计合成特异引物进行PCR扩增,获得了暗纹东方线粒体DNA控制区基因(818 bp)及5′端上游的tR-NAPro基因(71 bp)的全序列。控制区碱基组成为T32.2%,C 19.1%,A35.6%,G13.2%。对照其他已报道的鱼类控制区结构,对暗纹东方控制区的结构进行了分析,识别了其终止序列区、中央保守区和保守序列区,找到了终止相关的序列TAS以及保守序列(CSB1,CSB2,CSB3)。CSB1、CSB2序列相对保守,TAS与其回文基序可形成稳定的茎环结构,成为H-链复制延伸时的终止识别位点。同时运用DNA分析软件对暗纹东方与GenBank中其他10多种鱼类的mtDNA控制区序列进行比对,并选取东方属的7种鱼类mtDNA控制区序列构建分子系统树。结果显示控制区基因较适于科鱼类中同属不同种的系统发育分析。     

两种常见外来入侵赤潮藻的PCR鉴定

米氏凯伦藻与环状异帽藻为日本海域流入我国的两种外来入侵赤潮藻。利用核糖体ITS区分别设计出针对该两种藻的特异性PCR引物。通过prmier5.0软件设计多对引物,经PCR扩增,琼脂糖凝胶电泳检测、以筛选目标藻的特异性引物,并以链状亚历山大藻、立玛原甲藻、牟氏角毛藻、赤潮异弯藻作为阴性对照,做进一步PCR验证。筛选到米氏凯伦藻最佳引物Ki1F3/Ki1B3和环状异帽藻最佳引物YiF3a/YiB3a。两对特异性引物成功鉴定了两种外来入侵藻,而对其它藻种则是阴性反应,可为赤潮的预测预报提供分子鉴定基础。     

PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.