琥珀酸黄杆菌促生机理及其对多年生黑麦草生长和抗逆性的生理调控作用

为探究琥珀酸黄杆菌（Flavobacterium succinicans）对非生物胁迫下多年生黑麦草（Lolium perenne）的缓解效应,本试验研究了菌株DSM4002的生物学特性,并在盆栽条件下,分析了干旱（维持相对含水量（30±5）%处理7 d和14 d）、低温（4℃处理3 d和7 d）和盐胁迫（150 mM NaCl处理5 d和10 d）下根际接种DSM4002对多年生黑麦草生长和生理特性的影响。结果表明,DSM4002具有分泌吲哚乙酸（Indole-3-acetic acid,IAA）、溶磷和产铁载体能力,对壮观霉素、卡那霉素等具有抗性,对氯霉素敏感。同时,接种DSM4002能够提高正常生长、干旱、低温和盐胁迫下多年生黑麦草的生物量、抗氧化酶活性、可溶性糖和脯氨酸含量,降低相对电导率和丙二醛（Malondialdehyde,MDA）含量。综上,琥珀酸黄杆菌促进多年生黑麦草的生长与其自身具有分泌IAA、溶磷和产铁载体能力有关,而且它能够调节植物体内氧化还原平衡,从而增强多年生黑麦草的抗旱、抗寒和抗盐能力。     

Evaluation of a 4‐h static copper sulphate treatment against experimental infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus)

The efficacy of copper sulphate (CuSO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish (Ictalurus punctatus) was evaluated. Fish were challenged, by waterborne exposure to F. columnare in an ultra‐low flow‐through water delivery system, and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 at 5.5 h post challenge for 4 h. Bacterial challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged with F. columnare and treated with CuSO₄ at 4.2 and 2.1 mg L^?1 post challenge had mortalities of 6.4% and 18.3%, respectively, compared with the positive control, which had 90.2% mortality. The mortality of challenged fish treated with CuSO₄ at 4.2 and 2.1 mg L^?1 was significantly different from the positive control. There was no significant difference between the mortalities of the 4.2 and 2.1 mg L^?1 treatments. The results suggest that CuSO₄ has clear therapeutic value against columnaris infections.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Validation of diagnostic assays to screen broodstock for Flavobacterium psychrophilum infections

It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme‐linked immunosorbent assay (ELISA), a membrane‐filtration fluorescent antibody test (MF‐FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF‐FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron‐limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF‐FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub‐clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme.     

Diversity of Flavobacterium psychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids

Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome of salmonids. The pathogen has been reported from all regions in the world involved in salmonid aquaculture, but also from natural fresh-water environments. We established a quantitative loop-mediated isothermal amplification of DNA (LAMP) method to estimate quantities of F. psychrophilum . LAMP primers were designed based on the sequence of the DNA topoisomerase IV subunit B gene, parE , of F. psychrophilum. parE LAMP exhibited a high specificity for the parE gene of F. psychrophilum but not for other related species. parE LAMP detected the gene in a wide range of concentrations from 2.0 × 10¹ to 2.0 × 10⁹ copies/reaction within 70 min and revealed a good correlation between threshold times and gene copy number.     

柱状黄杆菌部分生物学特性的研究

采用革兰氏染色法、比色法和平板扩散法对柱状黄杆菌Flavobacterium columnare的部分生物学特性进行了研究。结果表明：用肉汤培养柱状黄杆菌24 h时,其形态多样,如半圆形、U型、V型或S型等;培养48 h时,柱状黄杆菌中开始出现颗粒状老龄培养物;培养72 h时,有部分菌体发生的＂分节＂现象,而老龄培养物也增多。用肉汤培养时,该菌的最佳生长条件分别是NaCl浓度为2.0 g/L,pH为7.4,温度为25～28℃。在最佳培养条件下,该菌的胞外产物具有蛋白酶、脂酶和卵磷脂酶活性,然而却检测不到淀粉酶、明胶酶和脲酶活性。     

柱状黄杆菌对草鱼TLRs基因表达水平的影响

用柱状黄杆菌Flavobacterium columnare G4株感染草鱼Ctenopharyngodon idella,分别在感染1、4、7d后提取感染组和对照组草鱼鳃、脾脏、肝脏、肠道和头肾5种组织的总RNA,并采用半定量RT-PCR方法检测不同组织中Toll样受体3(TLR3)、Toll样受体7(TLR7)和Toll样受体22(TLR22)3个抗病毒免疫基因的表达变化.结果表明:注射柱状黄杆菌7d后,TLR3在草鱼鳃、肝脏、肠道和头肾4种组织中的表达显著上调(P＜0.05);注射柱状黄杆菌4d和7d后,TLR7和TLR22在5种组织中的表达均显著上调(P＜0.05);而注射柱状黄杆菌1d后,TLR22在鳃、脾脏和肝脏组织中的相对表达量就显著上调(P＜0.05).研究表明,TLR3、TLR7和TLR22基因在机体应对细菌感染的免疫反应过程中发挥着重要的作用.