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.     

Detection of the bacterium Flavobacterium psychrophilum from a natural infection in rainbow trout, Oncorhynchus mykiss (Walbaum), using formalin-fixed, wax-embedded fish tissues

The bacterial pathogen Flavobacterium psychrophilum was successfully identified from formalin-fixed, wax-embedded tissue blocks of infected rainbow trout heart and spleen tissues, using a polymerase chain reaction (PCR)-based assay. Filamentous bacteria were observed in haematoxylin and eosin and Giemsa-stained sections but no bacteria were recovered from the diseased fish using standard bacteriology isolation techniques. All infected fish had histopathological evidence of myocarditis or rainbow trout fry syndrome. Immunohistochemistry was attempted using three different anti-F. psychrophilum sera but the results were inconclusive, and an alternative molecular approach was therefore attempted. This paper describes the use of a PCR-based assay to help identify bacteria present in formalin-fixed, wax-embedded tissue samples. This is the first time that this technique has been used for the detection of fish bacteria from diagnostic samples.     

Comparative proteomic analysis of virulent and rifampicin-attenuated Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild‐type strain (CSF 259‐93) with a rifampicin‐resistant strain and virulence‐attenuated strain of F. psychrophilum (CSF 259‐93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL‐43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL‐43. 2D‐PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole‐cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259‐93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259‐93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.     

患病异育银鲫中嗜冷黄杆菌的分离鉴定及组织病理学观察

为确定越冬期内江苏多地异育银鲫 (Carassiusauratus gibelio)暴发性死亡的原因,本研究利用高通量测序比较了健康样品和患病样品的微生物群落多样性和结构组成的差异,分析了异育银鲫病害暴发过程中的细菌类型及特性。结果表明：在属水平上,患病样品中黄杆菌属丰度最高。在种水平上,患病样品中嗜冷黄杆菌 (Flavobacterium psychrophilum)的丰度显著增加,分别达到63.01%和61.31%,显著高于健康组的1.55%（P < 0.05）。根据微生物群落特征分析结果,从患病样品体表的病灶处分离出优势病原菌NJ01,通过细菌形态学、生理生化分析、16S rDNA 序列比对确定 NJ01 菌株为嗜冷黄杆菌。人工感染NJ01菌株14d后,1.7×106～1.7×107 CFU/mL两组的死亡率达到100%,感染症状和自然发病症状一致。组织病理学观察发现,病鱼的肌细胞坏死,间质中充满了淋巴细胞;肝脏中细胞溶解坏死,细胞核消融;在脾脏中发现脾细胞散在坏死,伴随着充血的症状;肾小管上皮脱落,肾间质存在大量淋巴细胞。药物敏感试验结果表明,NJ01菌株对头孢西叮、头孢哌酮、庆大霉素和克拉霉素等敏感。本研究首次报道了嗜冷黄杆菌在异育银鲫中的致病性,这将为大宗淡水鱼“越冬综合征”的药物防治及其致病机理研究提供参考依据。     

Phenotypic and genotypic characterization of Flavobacterium psychrophilum from Finnish fish farms

Finnish isolates ( n =37) of Flavobacterium psychrophilum isolated from farmed salmonids were studied using phenotypic and genotypic characteristics. The characteristics of isolates were compared with the characteristics of Swedish and Estonian F. psychrophilum isolates and the type strain, F. psychrophilum NCIMB 1947^T. Multiple isolates from eight disease outbreaks were examined to determine differences between isolates from a single outbreak. The F. psychrophilum isolates represented a biochemically homogeneous group. However, some minor differences in biochemical and physiological characteristics were observed. Seven different antigenic patterns among Finnish isolates were detected and the results suggest a new serotype of F. psychrophilum . Using Cla Ι, Hae ΙΙΙ and Pvu ΙΙ restriction enzymes in ribotyping analyses 13 different genotypes were demonstrated and a possible relationship between serotype Fd and genotype F1 was determined. There were no significant differences between the isolates from Finland, Estonia, Sweden and the type strain of F. psychrophilum .     

Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout, Oncorhynchus mykiss (Walbaum), and spawning coho salmon, O. kisutch (Walbaum)

Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. This study examined the genetic diversity of F. psychrophilum isolates retrieved from multiple epizootics at rainbow trout, Oncorhynchus mykiss, rearing facilities and from spawning coho salmon, O. kisutch. A total of 139 isolates were confirmed as F. psychrophilum by PCR assay and were further typed using pulsed-field gel electrophoresis (PFGE). Multiple epizootics at three proximally located rainbow trout rearing facilities were numerically dominated by three PFGE profiles, which accounted for 76% of all trout isolates. In coho salmon, 19 PFGE profiles were differentiated by PFGE and four numerically dominant PFGE profiles represented 56% of all coho salmon isolates. PFGE analysis also indicated that the average similarity of macrorestriction patterns of F. psychrophilum isolates was greater in rainbow trout than in coho salmon (88% vs. 70%). Furthermore, it was not unusual to isolate multiple PFGE profiles from a single coho salmon sample whereas only two PFGE profiles were shared between two sample dates separated by 1 month. It is clear that the domestic rainbow trout aquaculture facilities studied here were primarily affected by a complex of genetically related strains whereas spawning coho salmon supported a much more genetically diverse collection of F. psychrophilum.     

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout,Onchorynchus mykiss (Walbaum), in Spain

Flavobacterium psychrophilum is the causative agent of bacterial cold‐water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed‐field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 μg mL^?1, and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.     

虹鳟源嗜冷黄杆菌的分离鉴定及致病性研究

为确定患病虹鳟的病原,本实验从患病鱼溃烂肌肉中分离到2株细菌,分别命名为CH06和CH07,经回归感染证实分离菌株为导致此次虹鳟患病的病原菌,并进一步对其形态特征、理化特性、分子特征、血清型及耐药性进行分析。结果显示,CH06和CH07株在TYES琼脂平板上呈煎蛋状外观,产黄色素,氧化酶和过氧化氢酶呈阳性,能水解明胶和酪蛋白,不能水解淀粉,不能利用果糖、半乳糖和七叶苷等。16S rRNA比对结果显示,CH06和CH07株与嗜冷黄杆菌模式株NBRC 15942的同源性分别为99.35%和99.42%。综合菌株理化和分子特性确定CH06和CH07株为嗜冷黄杆菌。利用多重PCR方法鉴定CH06和CH07株的血清型均为1型(Fd型);多位点序列分型(MLST)分析表明,CH06和CH07株的基因型分别为ST-12和ST-78型,且均属于CC-ST10克隆型。人工感染结果显示,CH06和CH07株对虹鳟幼鱼具有较高致病性,其半致死浓度(LD₅₀)分别为7.1×10⁵和1.1×10⁵ CFU/mL,攻毒剂量与临床病症出现时间呈反比,从人工感染实验鱼的肌肉、脾脏等组织中可重新分离到嗜冷黄杆菌。组织病理变化显示,病鱼肝细胞肿胀,空泡变性,部分肝细胞溶解坏死,细胞核溶解消失;脾脏充血、出血,淋巴细胞减少,红细胞和含铁血黄素增多;肌纤维间隙增宽、断裂、弯曲不齐,部分肌细胞肌浆溶解呈蜂窝状。CH06和CH07株对10种抗菌药物的耐药谱略有不同,均对氨苄西林和甲氧苄啶-磺胺甲噁唑敏感;CH06株对恩诺沙星、氟苯尼考等耐药,而CH07株对恩诺沙星和氟苯尼考中度敏感。本研究首次报道了我国虹鳟源嗜冷黄杆菌的分离鉴定及生物学特性,以期为虹鳟细菌性冷水病的防控提供科学依据。     

应用聚合酶链式反应(PCR)检测锦鲤疱疹病毒

为建立并优化锦鲤疱疹病毒检测方法,针对目前国内集约化养殖鲤鱼大面积流行的病死现象进行有针对性的检测,对在疫区采集到的病鱼肾脏、肝胰脏、肠等组织,提取基因组DNA,应用锦鲤疱疹病毒特异性引物进行PCR反应,同时设立阴性对照(TE buffer)。通过1%琼脂糖凝胶进行鉴定,与阴性对照比较,在484bp处出现特异性目的条带。经序列测定,所得PCR产物的基因序列与NCBI上锦鲤疱疹病毒的同源率达到99%以上。对同一批次的样品进行重复检测,结果表明该方法具有较好的可重复性,可据此判定结果。通过对锦鲤疱疹病毒的检测,确定了疫区引起框镜鲤(Cyprinus carpio)和建鲤(Cyprinus carpio var Jian)大批死亡的病原为锦鲤疱疹病毒(Koi Herpesvirus,KHV),从而对疫区控制、防治方案的制定提供了理论依据。建议尽快在鲤鱼主要养殖区开展KHV流行病学、诊断与检测及免疫预防等方面的研究。     

三疣梭子蟹病原弗氏柠檬酸杆菌的分离鉴定及定居因子抗原基因检测

2009年10月江苏赣榆地区某养殖场养殖的三疣梭子蟹出现大量死亡,症状主要表现为:病蟹行动缓慢、不摄食,蟹体消瘦,打开头胸甲可见肝胰腺、鳃、肌肉等内脏组织水肿,部分肝胰腺和肌肉组织呈腐烂状。从患病梭子蟹肌肉、肝胰脏、体内积液中分离到大量优势生长的细菌。人工感染试验,证明分离菌(JG091120-1)对健康三疣梭子蟹具有很强的致病性。对分离菌进行了形态特征、理化特性等常规表型生物学检验,同时利用分子生物学方法测定了代表菌株的16S rRNA和gyrB基因序列,其中分离菌16S rRNA基因序列长度为1 451 bp(登录号HQ170626),gyrB基因序列长度为1 186 bp(登录号HQ170627),分析了16S rRNA和gyrB两种基因序列的同源性。根据分离菌的表型及分子生物学特性,判定该菌为肠杆菌科枸橼酸属的弗氏柠檬酸杆菌。定居因子抗原cfa是肠杆菌科产肠毒素细菌的一种重要致病因子,利用特异性引物进行cfa基因的PCR扩增,分离菌可以扩增出大小在100 bp的基因片段,表明本次分离的病原弗氏柠檬酸杆菌具有cfa毒力因子。     

肝胰腺细小病毒和斑节对虾杆状病毒的PCR复合检测

肝胰腺细小病毒和斑节对虾杆状病毒是常见的2种DNA对虾病毒,在虾类养殖业中经常会出现混合感染。因此,建立这2种病毒的复合检测方法显得尤为重要。根据基因库中肝胰腺细小病毒和斑节对虾杆状病毒的保守序列,设计了肝胰腺细小病毒和斑节对虾杆状病毒的特异性引物,对这2种病毒PCR复合检测的反应条件和试剂浓度进行优化,退火温度55℃,主要试剂Mg2+终浓度为3mmol/L,dNTP终浓度为0.4 mmol/L,引物终浓度为0.8μmol/L。进一步比较了在优化后的PCR反应体系下,检测单种病毒和同时检测这2种病毒的灵敏度差异。     

异育银鲫病原温和气单胞菌表型及分子鉴定与溶血素基因检测

2008年10月江苏盐城大丰精养异育银鲫（Carassius auratus gibelio) 发生严重疾病,从病鱼肝脏、血液及腹水中分离到优势生长的细菌。对分离做纯培养的4株菌(JY081016-1至JY081016-4)进行形态特征、理化特性等表型生物学性状检验;测定了菌株（JY081016-1）的16S rRNA和gyrB基因序列,分析了16S rRNA和gyrB两种基因序列的同源性,并构建了系统发生树。结果表明分离菌对供试健康异育银鲫有强致病性,菌株（JY081016-1）所扩增的16S rRNA基因序列长度为1448bp（GenBank 登录号：GQ232759）,所扩增的gyrB基因序列长度为1177bp（GenBank 登录号：GQ232760）,其16S rRNA和gyrB基因序列与GenBank数据库中维氏气单胞菌和维氏气单胞菌温和生物型的16S rRNA和gyrB基因序列相似性均在97%以上;根据分离菌的表型及分子特征,判定分离鉴定的4株菌为温和气单胞菌（Aeromonas sobria）。胞外酶及溶血活性检测表明分离菌均能产生蛋白酶、卵磷脂酶、脂酶,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血;设计的特异性引物可扩增出溶血素基因。     

基于核酸适配体的PCR法检测溶藻弧菌及其灭活菌

溶藻弧菌(Vibrio alginolyticus)分布广,数量多,发病率高,是水产养殖中常见的条件致病菌,而对溶藻弧菌进行快速准确的识别鉴定是其病害防治的前提和基础。核酸适配体,因为具有较高的亲和特异性,在微生物的识别鉴定方面展现出了巨大的优势。本文利用核酸适配体和适配体筛选产物,通过结合、洗涤、加热分离、PCR扩增以及电泳检测等步骤,对溶藻弧菌进行了检测鉴定。结果表明,适配体和筛选产物都能对溶藻弧菌及其灭活菌进行较好的识别鉴定,适配体筛选产物对溶藻弧菌的检测下限为10~3cfu/mL,而对其灭活菌的检测下限为10~2cfu/mL,适配体对溶藻弧菌及其灭活菌的检测下限都可达到10 cfu/mL。该方法对溶藻弧菌有较好的亲和特异性,并能较好地区分溶藻弧菌与哈维氏弧菌等水产常见病原菌,在水产病害的检测中显示了较好的应用前景。     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

Sensitivity of seven PCRs for early detection of koi herpesvirus in experimentally infected carp,Cyprinus carpio L., by lethal and non-lethal sampling methods

Koi herpesvirus (KHV) causes an economically important, highly infectious disease in common carp and koi, Cyprinus carpio L. Since the occurrence of mass mortalities worldwide, highly specific and sensitive molecular diagnostic methods have been developed for KHV detection. The sensitivity and reliability of these assays have essentially focused at the detection of low viral DNA copy numbers during latent or persistent infections. However, the efficacy of these assays has not been investigated with regard to low-level viraemia during acute infection stages. This study was conducted to compare the sensitivity of seven different polymerase chain reaction (PCR) assays to detect KHV during the first hours and days post-infection (hpi; dpi), using lethal and non-lethal sampling methods. The results highlight the limitations of the assays for detecting virus during the first 4 dpi despite rapid mortality in experimentally infected carp. False-negative results were associated with time post-infection and the tissue sampled. Non-lethal sampling appears effective for KHV screening, with efficient detection in mucus samples obtained from external swabs during this early infection period (<5 dpi), while biopsies from gills and kidney were negative using the same PCR assays. Non-lethal sampling may improve the reliability of KHV detection in subclinical, acutely infected carp.     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

Detection of monodon baculovirus and white spot syndrome virus in apparently healthy Penaeus monodon postlarvae from India by polymerase chain reaction

The simultaneous presence of monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in apparently healthy postlarvae of Penaeus monodon from different hatcheries in India was studied by nested polymerase chain reaction (PCR). MBV could be detected in 54% of the samples. However, only 15% of samples were positive by non-nested reaction. WSSV could be detected in 75% of samples, 19% being positive by non-nested reaction. The results show simultaneous presence of WSSV and MBV in many samples at various degrees of infection. Only 14% of the samples analysed were negative for both viruses.