Morphological and genetic characteristics of Flavobacterium columnare isolates: correlations with virulence in fish

Variability in pathogenicity of Flavobacterium columnare makes disease treatment difficult because there is currently no way to easily recognize those strains that warrant aggressive treatments. In order to identify suitable virulence markers, 17 isolates of F. columnare were cultured from six different fish species. The DNA from all isolates was analysed using random amplified polymorphic DNA (RAPD). Bootstrap analysis of the RAPD data produced a tree with three major groups supported by bootstrap scores of 80-100%. Virulence of the isolates was determined by bath exposure of channel catfish, Ictaluruspunctatus (Rafinesque), and golden shiners, Notemigonus crysoleucas (Mitchill), to broth cultures of F. columnare. In channel catfish, 13 of 17 isolates produced 100% mortality within 48 h post-exposure. All isolates of cyprinid fish origin clustered in a single RAPD group. At least two of the four isolates that were not virulent in channel catfish were of cyprinid fish origin. There was a wide variation in cell morphology between isolates with lengths of cells or cell chains ranging from 3 to 11 microm, even under identical culture conditions. Most of the shorter or single cell isolates fell into a single RAPD group. No clear association was identified between virulence and any other characteristic, including RAPD group.     

A comparative study of Edwardsiella ictaluri parent (EILO) and E. ictaluri rifampicin-mutant (RE-33) isolates using lipopolysaccharides,outer membrane proteins,fatty acids,Biolog, API 20E and genomic analyses

The biological properties of Edwardsiella ictaluri RE-33 rifampicin-mutant and its parent strain EILO were analysed. RE-33 is an avirulent isolate used as a modified live vaccine against enteric septicaemia of catfish. Electrophoretic analysis of lipopolysaccharide (LPS) patterns showed high homology between both isolates. Further characterization of LPS by immunoblotting revealed the main differences in LPS composition. The RE-33 isolate lacks the high molecular weight bands of LPS (HMW-LPS). Outer membrane protein analysis also showed some immunological differences between RE-33 and the EILO parent strain. Only two fingerprinting techniques, fatty acid composition analysis and Biolog phenotypic profiles, were able to discriminate between both isolates.     

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.     

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.     

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.     

Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of practical treatments for warmwater aquaculture ponds

Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare. In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h. Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F. columnare. Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero. With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033). Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective. Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish. Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.     

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。     

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.     

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.     

Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), hatcheries: studies on broodstock, eggs, fry and environment

The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.     

Biochemical and Serological Characterization of Flavobacterium columnare from Freshwater Fishes of Eastern India

Flavobacterium columnare is an important pathogen of freshwater fishes often causing high mortality. Seven strains of F. columnare have been isolated from gill necrosis, skin lesions and internal organs of Catla catla, Labeo rohita, Cirrhinus mrigala, Carassius auratus, Anabas testudineus and Clarias batrachus and characterized by biochemical reactions and serological tests viz indirect enzyme‐linked immunosorbent assay (ELISA), Dot‐ELISA, agglutination test. All the strains showed binding to Congo red dye as well as hemolysis. All the seven strains were susceptible to amikacin, gentamycin and ofloxacin. In vivo LD₅₀ dose of virulent strain MS2 was found to be 6 × 10⁴ CFU/mL after experimental infection to L. rohita. Serologically all the seven strains showed a positive result to agglutination, Dot‐ELISA and indirect ELISA. The agglutination titer was found to be in the range of 256‐131,072. The lysozyme activity of hyperimmune sera was found to be 39.37 ± 0.80 units/mL. This is the first extensive study that report about the various strains of F. columnare from freshwater fishes of Eastern India and their differentiation by biochemical and serological methods.     

Isolation and characterization of Flavobacterium columnare strains infecting fishes inhabiting the Laurentian Great Lakes basin

Flavobacterium columnare, the aetiological agent of columnaris disease, causes significant losses in fish worldwide. In this study, the prevalence of F. columnare infection was assessed in representative Great Lakes fish species. Over 2000 wild, feral and hatchery‐propagated salmonids, percids, centrarchids, esocids and cyprinids were examined for systemic F. columnare infections. Logistic regression analyses showed that the prevalence of F. columnare infection varied temporally and by the sex of the fish, whereby females had significantly higher prevalence of infection. A total of 305 isolates of F. columnare were recovered. Amplification of the near complete 16S rRNA gene from 34 representative isolates and subsequent restriction fragment length polymorphism analyses demonstrated that all belonged to F. columnare genomovar I. Phylogenetic analysis of near complete 16S rRNA gene sequences also placed the isolates in genomovar I, but revealed some intragenomovar heterogeneity. Together, these results suggest that F. columnare genomovar I is widespread in the Great Lakes Basin, where its presence may lead to mortality.     

Evaluation of diquat against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L⁻¹ of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L⁻¹ significantly ( P  < 0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro , the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 μg mL⁻¹ (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection.     

Molecular characterization of Portuguese strains of Yersinia ruckeri isolated from fish culture systems

A total of 23 Portuguese strains of Yersinia ruckeri , the causative agent of enteric redmouth disease (ERM), were comparatively studied by means of lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, plasmid profiling and ribotyping in order to investigate the heterogeneity among isolates and the usefulness of these methods as epidemiological markers. Only two LPS profiles were observed among the isolates studied, corresponding with the serotypes O1 and O3 of Y. ruckeri . A higher heterogeneity was detected analysing the OMP, seven different patterns being observed. Although some isolates carried different small plasmids, all the serotype O1 isolates showed a plasmid band of 50 MDa and all the serotype O3 strains shared in common a extrachromosomic DNA band of 30 MDa. The analysis of the ribopatterns obtained using three different enzymes, separated the strains into 10 ribotypes, indicating genetic heterogeneity among the isolates. The heterogeneity was greater within serotype O1 isolates, six ribotypes being detected, than within serotype O3 in which only three ribotypes were found. Although OMP analysis and ribotyping can be useful for the differentiation of strains on the basis of farm and/or season of isolation, we consider that ribotyping is the best candidate for epidemiological studies because it is easier to perform and offers a slightly better discriminative power.     

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...     

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.     

Feed deprivation of channel catfish, Ictalurus punctatus (Rafinesque), influences organosomatic indices, chemical composition and susceptibility to Flavobacterium columnare

Withholding feed has been suggested as a strategy to manage infectious disease of channel catfish, Ictalurus punctatus (Rafinesque). In a previous study, we demonstrated that deprivation of feed for as little as 7 days reduced innate resistance of catfish to Flavobacterium columnare. This study was conducted to evaluate the effect of feeding regimens [no feeding (NF), fed once every other day to satiation (FEOD) and fed once daily to satiation (FD)] on organosomatic indices, physiological changes and susceptibility of channel catfish to F. columnare. Fish that were not fed for 2 and 4 weeks had a significant increase (P < 0.05) in gutted weight:-wet weight ratio and decrease in other organosomatic indices [gut index (GI), mesenteric fat index (MFI) and hepatosomatic index (HSI)]. Haematology was not effected by feeding regimen except at week 4, when a significantly higher haemoglobin level was observed in the NF fish. Serum protein did not differ at week 2, but the level at week 4 of the NF fish (35.91 mg mL(-1)) was significantly lower than that of the FD fish (41.77 mg mL(-1)). Significantly lower (P < 0.05) blood glucose (39.5 and 40.3 mg dL(-1)) and liver glycogen (1.7 and 1.8 mg g(-1)) were seen in the NF fish at weeks 2 and 4, respectively, as compared with blood glucose and liver glycogen levels of FD fish (67.5 and 92.8 mg dL(-1) and 46.5 and 52.6 mg g(-1) at weeks 2 and 4, respectively) and FEOD (82.8 and 85.5 mg dL(-1) and 45.1 and 51.4 mg g(-1) at weeks 2 and 4, respectively). Mortality in the NF fish caused by F. columnare (78%) was significantly higher (P < 0.05) than mortality in the FD and FEOD treatments (0.0 and 1.7%, respectively). Blood glucose and liver glycogen showed the same trend of low values for NF fish following challenge (week 6). Blood glucose, liver glycogen, GI and HSI are sensitive indicators for channel catfish deprived of feed (NF) for 4 weeks. Blood glucose and liver glycogen levels around 40 mg dL(-1) and 2 mg g(-1), respectively, are indicative of starvation in juvenile channel catfish. Moreover, NF fish were susceptible to F. columnare infection. Thus, it is suggested that in the absence of natural food, juvenile channel catfish should be fed at least once every other day to apparent satiation to maintain normal physiological function and improve resistance to F. columnare.     

Influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum)

The influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied experimentally in the laboratory and at a fish farm. In experiment I, the effect of parasitic infection on columnaris disease was studied using F. columnare carrier fish. The fish were exposed to Diplostomum spathaceum cercariae and a set of other stressors in order to induce clinical columnaris infection. Parasitic infection and other stressors failed to induce the disease. Disease occurred when the fish were challenged with F. columnare, but D. spathaceum infection did not enhance the severity of the infection. In experiment II, the influence of rearing density and water temperature was studied. Overall mortality was highest in fish at normal rearing density with high temperature (+23 degrees C). At low temperature (+18 degrees C) mortality was not affected by rearing density, but the transmission of columnaris disease was faster at normal rearing density at both temperatures. This supports the view that reduction of fish density could be used in prevention of columnaris disease especially if water temperature is high. Because the lower rearing density can also decrease the transmission of ectoparasites and penetrating endoparasites, it could be an efficient tool in ecological disease management.