Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare

The virulence of eight Flavobacterium columnare strains was studied to find correlations between several virulence-related factors and virulence. Virulence was tested in vivo using rainbow trout, Oncorhynchus mykiss (Walbaum). Suggested virulence-related factors such as production of the degradative enzyme chondroitin lyase, plasmid occurrence and adhesion capability were studied in vitro. Infection with the four most virulent strains resulted in 95-100% mortality within 114 h. Chondroitin lyase activity was found to be significantly related to the virulence of the strains at 25 degrees C and it was also shown to be temperature-dependent, being higher at 25 degrees C than at 20 degrees C. Virulence was not plasmid associated. The adhesion capability of the strains in vitro varied substantially when tested on crude mucus-coated slides and no statistical relationship between adhesion and virulence was found using this method.     

Difference in genes between a high virulence strain G4 and a low virulence strain G18 of Flavobacterium columnare by using suppression subtractive hybridization

Flavobacterium columnare is the causative agent of columnaris disease. Different genetic groups of F. columnare show to some extent different degrees of virulence. To identify genetic differences between the high virulence strain G₄ and the low virulence strain G₁₈ of F. columnare, suppression subtractive hybridization was used. A total of 46 genes were identified from the virulent strain G₄, 35 of which showed some degree of homology with known proteins and can be classified into 11 categories: DNA replication or recombination proteins, inorganic ion transport proteins, outer membrane proteins, enterotoxin, binding proteins, YD repeat proteins, transposase, chaperon, signal transduction‐related proteins, regulatory proteins, metabolism‐related proteins. Several putative virulence factors identified in other bacteria could also be identified in the virulent strain G₄, such as ferrous iron transport protein, TonB‐dependent receptor, transposases, as well as ABC transporter permease protein. The flanking region of a putative transposase ISFclI was analysed, and a putative Rhs element was located at the downstream of the putative transposase. The analysis of isfclI gene in 24 strains of F. columnare isolated in China revealed that 11 strains have isfclI, and all the strains from Zhaoqing, Anhui and Qingjiang have isfclI.     

Lack of association between Flavobacterium columnare genomovar and virulence in hybrid tilapia Oreochromis niloticus (L.) × Oreochromis aureus (Steindachner)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex‐reversed hybrid tilapia, Oreochromis niloticus (L.) × O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II‐B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3–78 and 3.3–75%, respectively. The mean per cent mortality of fish challenged with genomovar II‐B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host.     

Effects of low salinities on Flavobacterium columnare infection of euryhaline and freshwater stenohaline fish

Channel catfish, Ictalurus punctatus (Rafinesque), goldfish, Carassius auratus (L.), striped bass, Morone saxatilis (Walbaum), and Gulf sturgeon, Acipenser oxyrinchus desotoi Vladykov, were acclimatized to fresh water or salinities of 9.0‰ or less and then exposed to Flavobacterium columnare (formerly known as Flexibacter columnaris ), the bacterial pathogen that causes columnaris disease. None of the fish acclimatized to 3.0 or 9.0‰ salinity died, and all deaths in lower salinities occurred between 1 and 5 days after exposure to F. columnare . Mortality was 97.7% in fresh water and 67.1% in 1.0‰ salinity for channel catfish (model SE, 1.8) and 66.5% in fresh water and 40.8% in 1.0‰ salinity for goldfish (model SE, 1.2); and 96.9% in fresh water and 61.7% in 1.0‰ salinity for striped bass (model SE, 1.8). After exposure to F. columnare , none of the Gulf sturgeon died. Flavobacterium columnare was isolated from the skin and gills of all fish dying during the experiments, but was not isolated from survivors in fresh water and 1.0‰ salinity 21 days after bacterial exposure. In vitro growth of bacteria was significantly higher in 1.0 or 3.0‰ salinity than in control medium (0.3‰ salinity). However, in vitro adhesion of bacteria was reduced with increasing salinity, which could explain the lower mortality of fish at higher salinities.     

柱状黄杆菌强毒株和弱毒株胞外蛋白中差异蛋白的初步鉴定

柱状黄杆菌(Flavobacterium columnare)是一种世界范围的水产动物致病菌,是中国重要养殖鱼类草鱼(Ctenopharyngodon idellus)、鳜(Siniperca chuatsi)等烂鳃病的病原。本研究以1972年从患"烂鳃病"草鱼上分离的两株冻干柱状黄杆菌G4和G18菌株为研究对象,并将G4株再次分离纯化得纯化菌株,命名为G4R3。对草鱼鱼苗浸泡攻毒结果显示,G4R3的LD50至少比G18的高3个数量级,因此G4R3为"强毒株",G18为"弱毒株"。利用蛋白质组学方法分析柱状黄杆菌强毒株G4R3和弱毒株G18的胞外蛋白,经过双向电泳并结合图像分析,共发现了34个点是差异表达的蛋白。胶内酶解、肽质量指纹图谱和串联质谱分析后,鉴定出其中的7个蛋白点,代表滑动蛋白K、腺酐甲硫氨酸合成酶和一种可能的膜蛋白等3种蛋白,它们可能是柱状黄杆菌的毒力因子。     

Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.     

Comparison of lipopolysaccharide and protein profiles between Flavobacterium columnare strains from different genomovars

Lipopolysaccharide (LPS) and total protein profiles from four Flavobacterium columnare isolates were compar. These strains belonged to genetically different groups and/or presented distinct virulence properties. Flavobacterium columnare isolates ALG-00-530 and ARS-1 are highly virulent strains that belong to different genomovars while F. columnare FC-RR is an attenuated mutant used as a live vaccine against F. columnare. Strain ALG-03-063 is included in the same genomovar group as FC-RR and presents a similar genomic fingerprint. Electrophoresis of LPS showed qualitative differences among the four strains. Further analysis of LPS by immunoblotting revealed that the avirulent mutant lacks the higher molecular bands in the LPS. Total protein analysis displayed by immunoblotting showed differences between the strains analysed although common bands were present in all the isolates. FC-RR lacked two distinct common bands (34 and 33 kDa) shared by the other three isolates. Based on the difference of LPS and total protein profiles, it is possible to discriminate the attenuated mutant FC-RR from other F. columnare strains.     

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.     

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L‐rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L‐rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L‐rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L‐rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.     

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 characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac‐Col^®. The strain of F. columnare used to generate the avirulent rifampicin‐resistant mutant used in Aquavac‐Col^® belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin‐resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin‐resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid

Commercial products containing peracetic acid (PAA) are strong disinfectants with a wide spectrum of antimicrobial activity and have been suggested as potential therapeutic agents in aquaculture. The aim of this study was to compare the in vitro reduction of growth on two fish pathogens, Flavobacterium columnare and Saprolegnia parasitica, by seven commercial PAA‐containing products. Flavobacterium columnare was exposed to 1, 2, 4, 6, 8 and 10 mg L^?1 PAA and S. parasitica was exposed to 0.5, 1, 2, 4, 6, 8 and 10 mg L^?1 PAA in petri dishes for 24 h incubation. The reduction of growth was measured in comparison to a PAA‐free control. A reduction of the growth was observed for both pathogens with increasing PAA concentration. Hydrogen peroxide (H₂O₂) possibly has a role in the effectiveness of the products, since products with lower PAA concentrations had a higher concentration of H₂O₂. The commercial products with a low concentration of PAA and a low PAA:H₂O₂‐ratio were generally more effective against pathogens. The practical application of the products with low PAA concentration should be prioritized.     

Isolation and characterization of strains of Flavobacterium columnare from Brazil

Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare.     

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Flavobacterium columnare

Outer membrane proteins (OMPs) of bacteria are key molecules interacting with the host environment. Flavobacterium columnare, a pathogen-causing columnaris disease of fish worldwide, was studied in order to understand the composition of its OMPs. The sarcosine-insoluble membrane fraction of the OMPs was analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with reverse-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC MS/MS). Thirty-six proteins were identified, including proteins involved in cell wall/membrane biogenesis, specific transport of various nutrients and in essential metabolism. The present study is the first report on the OMPs of F. columnare, and may serve as the basis for understanding the pathogenesis of the bacterium.     

Kaolinitic clay protects against Flavobacterium columnare infection in channel catfish Ictalurus punctatus (Rafinesque)

Columnaris disease, caused by the bacterial pathogen Flavobacterium columnare, continues to be a major problem worldwide in both wild and cultured freshwater finfish. Despite the far-reaching negative impacts of columnaris disease, safe and efficacious preventatives and curatives for this disease remain limited. In this study, we evaluated the potential of kaolin (Al₂Si₂0₅(OH)₄), a type of clay, for the prevention of columnaris disease. Channel catfish, Ictalurus punctatus (Rafinesque), fingerlings were experimentally challenged with Flavobacterium columnare in untreated water or with water containing kaolin (1 g L⁻¹). Over the 7-day course of study, kaolin treatment led to significantly (P < 0.001) improved survival (96%) as compared to untreated fish (78% survival). Histological examination of the gills revealed that kaolin-treated fish had substantially less gill damage than untreated controls. Quantitative PCR analysis of gill tissue revealed that kaolin significantly reduced F. columnare adhesion (measured at 1 h post-challenge) and colonization (24 h post-challenge). Incubation of kaolin with F. columnare in vitro demonstrated that kaolin reduced the number of F. columnare cells in culture supernatants, presumably through the formation of physical complexes through adsorption. In summary, kaolin can improve survival, reduce gill pathologies and reduce bacterial attachment to key tissues associated with columnaris disease in channel catfish by binding to F. columnare.     

柱状黄杆菌乙酰辅酶A合成酶基因及其上游调控序列分析

柱状黄杆菌(Flavobacterium columnare)是世界范围内危害淡水鱼类的柱形病的病原。目前对该病原菌遗传操作系统的研究进展较慢,而寻找高效稳定的启动子来调控外源基因在细菌体内的表达,有可能促进该细菌遗传操作系统的构建。本研究获得了柱状黄杆菌乙酰辅酶A合成酶基因(acetyl-coenzyme A synthetase gene,acs)的编码序列及其上游调控序列,该基因全长2 323 bp,编码635个氨基酸。通过序列分析,发现在该基因起始密码子ATG的上游存在核糖体结合位点(ribosome biding site,RBS)序列TAAAA,和启动子–7和–33的保守基序TATTTTCG和TTG。将acs的上游调控序列(promoter sequence,Pacs)置于氯霉素抗性基因(chloramphenicol acetyltransferase,cat)的上游并导入柱状黄杆菌G4株后,cat基因得以表达,并使宿主细胞产生稳定的氯霉素抗性。通过5′RACE技术,确定了外源的cat基因和内源的acs基因的转录起始位点都是位于起始密码子上游46 bp处的T。通过删减分析调控序列Pacs,发现起...