Search

This study was conducted to determine antibiotic susceptibility of Flavobacterium psychrophilum isolated in diseased rainbow trout (Oncorhynchus mykiss) from two hatcheries in the Mediterranean region of Turkey. The samples were taken from the kidney and spleen for bacteriology from rainbow trout juveniles (weight 0.7-1 g) in April and isolates obtained on tryptone yeast extract salts (TYES) agar medium at 15°C for 72 hours. Fourteen isolates were identified as F. psychrophilum using biochemical, physiological, and morphological characteristics and API ZYM test. Antibiotic sensitivities of the isolates were determined by the disc diffusion method on TYES agar, and the results were evaluated according to CLSI standards. Only one isolate from each hatchery was used for the antibiogram test. Both isolates were sensitive to amoxicillin/clavulanic acid 2:1, penicillin, nitrofurantoin, florfenicol, amoxicillin, clindamycin, chloramphenicol, ampicillin, doxycycline, streptomycin, and erythromycin; resistant to sulfadiazine, sulfamethoxazole/trimethoprim, oxolinic acid, vancomycin, trimethoprim/sulfadiazine, gentamicin, kanamycin, nalidixic acid, oxacillin, enrofloxacin, flumequine, tobramycin. The results show that isolating F. psychrophilum and performing the antimicrobial susceptibility testing is important issue in disease control.

Background: Bacteriophages (phages) have been proposed as an alternative to antibiotics and surface disinfectants for treatment of Flavobacterium psychrophilum biofilms and fish infections in aquaculture settings. The aim of the study was to estimate the minimal phage:host ratio (PHR) required for the control of in vitro biofilm formation and mortalities caused by F. psychrophilum in experimentally infected fish. Materials and Methods: F. psychrophilum cells in different stages of biofilm formation were exposed to the lytic phage FPSV-D22 at different PHRs. Results: Our results show that an initial PHR of 0.01 is sufficient for more than an 80% inhibition of attachment and colonization, and disruption of maturated F. psychrophilum biofilms, whereas greater ratios resulted in almost complete interruption of the different biofilm stages. Interestingly, a similar response was observed in a phage therapy trial with live rainbow trout (Oncorhynchus mykiss), where treatment of F. psychrophilum-infected fish by injection of serial bacteriophage doses resulted in significantly (***p ≤ 0.001) higher survival already at a PHR of 0.02. Conclusions: These results indicate that phages have the potential to be effective for control and treatment of F. psychrophilum infections in fish farms even when applied in concentrations lower than previously expected.

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, twenty-seven phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence, and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage resistant phenotypes.

BackgroundRadiation-associated angiosarcoma of the breast (RAASB) is an aggressive malignancy that is increasing in incidence. Only a few previous population-based studies have reported the results of RAASB treatment.MethodsA search for RAASB patients was carried out in the Finnish Cancer Registry, and treatment data were collected to identify prognostic factors for survival.ResultsOverall, 50 RAASB patients were identified. The median follow-up time was 5.4 years (range 0.4–15.6), and the 5-year overall survival rate was 69%. Forty-seven (94%) patients were operated on with curative intent. Among these patients, the 5-year local recurrence-free survival, distant recurrence-free survival, and overall survival rates were 62%, 75%, and 74%, respectively. A larger planned surgical margin was associated with improved survival.ConclusionsWe found that the majority of RAASB patients were eligible for radical surgical management in this population-based analysis. With radical surgery, the prognosis is relatively good.

Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

Background: Bacteriophages (phages) have been proposed as an alternative to antibiotics and surface disinfectants for treatment of Flavobacterium psychrophilum biofilms and fish infections in aquaculture settings. The aim of the study was to estimate the minimal phage:host ratio (PHR) required for the control of in vitro biofilm formation and mortalities caused by F. psychrophilum in experimentally infected fish. Materials and Methods: F. psychrophilum cells in different stages of biofilm formation were exposed to the lytic phage FPSV-D22 at different PHRs. Results: Our results show that an initial PHR of 0.01 is sufficient for more than an 80% inhibition of attachment and colonization, and disruption of maturated F. psychrophilum biofilms, whereas greater ratios resulted in almost complete interruption of the different biofilm stages. Interestingly, a similar response was observed in a phage therapy trial with live rainbow trout (Oncorhynchus mykiss), where treatment of F. psychrophilum-infected fish by injection of serial bacteriophage doses resulted in significantly (***p ≤ 0.001) higher survival already at a PHR of 0.02. Conclusions: These results indicate that phages have the potential to be effective for control and treatment of F. psychrophilum infections in fish farms even when applied in concentrations lower than previously expected.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g–1) or by irreversible immobilization (8.3 × 107 PFU g–1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg–1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml–1; 3rd bath: ∼105 PFU ml–1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish–1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. psychrophilum infections.

The fish pathogen Flavobacterium psychrophilum is currently one of the main pathogenic bacteria hampering the productivity of salmonid farming worldwide. Although putative virulence determinants have been identified, the genetic basis for variation in virulence of F. psychrophilum is not fully understood. In this study, we analyzed whole-genome sequences of a collection of 25 F. psychrophilum isolates from Baltic Sea countries and compared genomic information with a previous determination of their virulence in juvenile rainbow trout. The results revealed a conserved population of F. psychrophilum that were consistently present across the Baltic Sea countries, with no clear association between genomic repertoire, phylogenomic, or gene distribution and virulence traits. However, analysis of the entire genome of four F. psychrophilum isolates by hybrid assembly provided an unprecedented resolution for discriminating even highly related isolates. The results showed that isolates with different virulence phenotypes harbored genetic variances on a number of consecutive leucine-rich repeat (LRR) proteins, repetitive motifs in gliding motility-associated protein, and the insertion of transposable elements into intergenic and genic regions. Thus, these findings provide novel insights into the genetic variation of these elements and their putative role in the modulation of F. psychrophilum virulence.

Increasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence.ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

The bacterium Pseudomonas anguilliseptica has in recent years emerged as a serious threat to production of lumpfish in Norway. Little is known about the population structure of this bacterium despite its association with disease in a wide range of different fish species throughout the world. The phylogenetic relationships between 53 isolates, primarily derived from diseased lumpfish, but including a number of reference strains from diverse geographical origins and fish species, were reconstructed by Multi-Locus Sequence Analysis (MLSA) using nine housekeeping genes (rpoB, atpD, gyrB, rpoD, ileS, aroE, carA, glnS and recA). MLSA revealed a high degree of relatedness between the studied isolates, altough the seven genotypes identified formed three main phylogenetic lineages. While four genotypes were identified amongst Norwegian lumpfish isolates, a single genotype dominated, irrespective of geographic origin. This suggests the existence of a dominant genotype associated with disease in production of lumpfish in Norwegian aquaculture. Elucidation of the population structure of the bacterium has provided valuable information for potential future vaccine development.

Rainbow trout fry syndrome (RTFS) is an important infectious disease caused by Flavobacterium psychrophilum affecting farmed salmonids worldwide. Other Flavobacterium psychrophilum-like species (F. plurextorum, F. oncorhynchi, F. tructae, F. collinsii and F. piscis) have been isolated from diseased rainbow trout fry suspected of RTFS although the epidemiological and clinical relevance of these pathogens are unknown. The objective of this study was to evaluate the potential use of MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight) Mass Spectrometry as method for specific identification of F. psychrophilum and its differentiation from other F. psychrophilum-like species isolated from diseased fish. Fifty-three isolates were analyzed after the creation of the Main Spectrum Profile (MSP) of reference strains of each of abovementioned species. F. psychrophilum exhibited a mass spectra very different from those of F. psychrophilum-like species, with five peaks (m/z 3654, 4585, 5388, 6730 and 7310) present only in F. psychrophilum isolates, and three peaks (m/z 6170, 7098 and 9241) absent in F. psychrophilum but present in all F. psychrophilum-like species. All F. psychrophilum isolates were correctly identified and differentiated from the F. psychrophilum-like species by MALDI-TOF. Although this approach showed a limited ability to differentiate among F. psychrophilum-like species, its complementation with a few simple biochemical tests may represent an alternative approach for the routine identification of the Flavobacterium psychrophilum-like species.

In this study, we isolated Candida sp. for the first time from fry of rainbow trout ( Oncorhynchus mykiss ) in Turkey. Candida species caused disease in fry (average 0.2 g) which was observed at 10°C water temperature in February 2016. Due to yeast accumulation on gills, whitish areas was seen and fusion of secondary lamella in gills were observed under microscope. The microbiological samples of kidney, spleen and gills were streaked onto trypticase soy agar (TSA) and tryptone yeast extract salts ( TYES ) agar plates and incubated at 22°C in TSA and 18°C in TYES agar for 48 h. Thirteen isolates were obtained and Gram staining was performed. The all of the isolates were determined as yeast cell based on cellular morphology by using light microscopy . The identification was performed by ITS (Internal transcribed spacer) region sequence analysis and sequence data demonstrated that representative isolate D11 belonged to genus Candida . The yeast infection might have seen in early development stages (larvae and fry) due to the intensive antibiotic use against rainbow trout fry syndrome (RTFS) disease.

Bacterial pathogens, preliminary identified as Edwardsiella sp., were isolated from farmed whitefish, Coregonus lavaretus (L.), in Finland during disease outbreaks in 2000, 2002 and 2013. The diseased fish exhibited clinical and histopathological signs of general septicemia. Bacterial diagnostics were initially performed with conventional biochemical tests and the API50 CH rapid diagnostic system, followed by a species-specific PCR assay. Antimicrobial susceptibility profiles, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), multilocus sequence analysis (MLSA) and biofilm formation were used to characterize the bacterial isolates. For comparison, two reference strains Edwardsiella piscicida ET883T (NCIMB 14824) and Edwardsiella tarda ATCC 15947T were included in the analyses. Biochemical and antimicrobial analysis displayed the same profile for all isolates from whitefish and this was similar to both reference strains. Molecular assays including PCR and ERIC-PCR demonstrated that all whitefish isolates were genetically different from the E. tarda reference strain. MLSA analysis revealed that the whitefish isolates and ET883T are in a separate clade distinct from ATCC 15947T; they are not genetically identical. It is concluded that whitefish is susceptible to infection with E. piscicida a bacterial species with a wide geographical distribution and adaptability to different hosts. It is postulated that in the future E. piscicida might pose a serious threat to farmed whitefish especially in recirculating aquaculture systems. Statement of relevance Edwardsiella piscicida was recently described and separated from the species Edwardsiella tarda. E. tarda/piscicida is a well-known fish pathogen infecting mainly warm water species. However, during recent years the pathogen has also been isolated from cold water fish species, mainly salmonids. It is not known if this is due to an adaptation of the pathogen to lower temperatures or due to farming of these fish in higher temperatures. In the present study E. piscicida was isolated and described for the first time from disease outbreaks of whitefish (salmonid species) in different freshwater farms in Finland. Farming of whitefish is increasing in Finland, especially in recirculating systems. The examined isolates were all obtained from farmed whitefish and it is hypothesized that this pathogen could be an increasing threat for the future farming of whitefish.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15–23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts., Sub-lineages of the fish-pathogenic bacterium Aeromonas salmonicida display specific host preferences.

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in farmed rainbow trout (Oncorhynchus mykiss), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of F psychrophilum, or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD50) of 26 geographically disparate F psychrophilum isolates was determined in rainbow trout. Furthermore, the in vitro sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested F psychrophilum isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD50 (8.9 x 103 to 3.1 x 106 CFU). No association between MLST ST and virulence was found because of a high variation in LD50 within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD50 5 CFU), moderate (LD50 = 105-106 CFU), and weak (LD50 > 106 CFU) virulence were similar in phenotypic characteristics in vitro. However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in F. psychrophilum. Univariate correlation studies found only non-significant associations between LD50 and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic F. psychrophilum strains should be further explored.

Turkey was the largest rainbow trout producer of the European countries in 2016, and the reason for this production is mainly attributed to its egg and fry production. Flavobacterium psychrophilum cause the highest rates of mortality in the starting to feeding stages of the fish. In the present study, twenty-five F. psychrophilum isolates recovered from rainbow trout, coruh trout and brook trout were analysed by RAPD-PCR, ERIC-PCR, REP-PCR and PCR-RFLP, including 16S rRNA, gyrA and gyrB gene regions and PCR-based serotyping method. The PCR-based molecular analysis showed that the isolates could not be differentiated exactly according to isolation source and geographical region. Most isolates were of type-1 and type-2, and some of them were of type-0 and type-3; in addition, one isolate showed a unique serotype. The combined analysis results showed that F. psychrophilum isolates discriminated as five different genotypes and all isolates were successfully discriminated based on host.

To investigate whether European whitefish (Coregonus lavaretus) groups in the brackish (≤7%) Gulf of Bothnia (Baltic Sea) express specific otolith elemental characteristics, concentrations of elements (Ca, Ba, Sr, Zn, Mn, Fe) in whole otoliths were studied by inductively coupled plasma-optical emission spectrometry. Whitefish (N = 67) were sampled from six different latitudinal sites; four in the sea and two in rivers along the west coast of Finland, and from a fresh water lake for comparison. The concentration of several elements showed significant differences among sampling sites. Otolith Ba concentrations were higher at the northern sampling sites compared to the southern ones, thereby showing negative association to capture-habitat salinity. In contrast, otolith Sr and Zn concentrations were lower at the northern sampling sites, thereby associating positively to capture-habitat salinity. Otolith Mn concentrations did not associate with latitude or salinity but markedly varied between sampling sites. Elevated Fe concentrations occurred in fish otoliths from the two northernmost sites. Ca concentrations were at the same levels in otoliths from all whitefish samples. Otoliths of whitefish from the lake had very high Ba and low Sr concentrations compared to concentrations in otoliths from whitefish in the sea and rivers. Our results show that otolith elemental characteristics of whitefish vary considerably at various geographic scales showing high potential for population identification using elemental fingerprints. Analysis of otolith elemental characteristics may therefore provide an efficient tool for the identification of main areas of residence, spawning locations, and migration routes, thus contributing towards the sustainable management of whitefish stocks.

Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

Phenotypic smooth cells of the fish pathogenic bacterium Flavobacterium psychrophilum have previously been reported to be more adhesive to polystyrene surfaces than corresponding rough cells. In this study, the adhesion ability of smooth and rough cells of F. psychrophilum to polystyrene surfaces was investigated in detail with a crystal violet staining method. By treating both polystyrene surfaces with fish mucus and carbohydrates and the bacterial cells with carbohydrates, the involvement of lectins in the adhesion process was investigated. Smooth cells showed significantly higher adhesion ability to untreated polystyrene surfaces compared with corresponding rough cells and increasing water hardness had an inhibitory effect on the adhesion. Treatment of polystyrene surfaces with D-glucose, D-galactose and fish mucus increased the adhesion ability of smooth cells to polystyrene. Furthermore, treatment of the smooth cells with D-glucose, D-galactose and sialic acid decreased the adhesion ability of the cells, indicating that the adhesion is likely mediated by complementary lectins on the surface of the cells. Sodium (meta)periodate treatment of smooth cells also decreased the adhesion ability to polystyrene, suggesting that the lectins, such as the dominating sialic acid-binding lectin, are probably localized in the extracellular polysaccharides surrounding the cells.

The antimicrobial susceptibilities of TurkishF. psychrophilumisolates were investigated using a Clinical and Laboratory Standards Institute (CLSI) standard microdilution testing protocol. The significance of the minimum inhibitory concentrations was determined by applying empirically and statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Twenty-fiveF. psychrophilumisolates made over four years from three different trout species in five different regions were examined. All isolates were fully susceptible to amoxicillin and florfenicol, but 88% of our isolates were categorized as having reduced susceptibility to the two quinolones, oxolinic acid, and enrofloxacin. Eighty percent of the isolates were categorized as showing reduced susceptibility to oxytetracycline.Sequence analysis demonstrated that the reduced susceptibility to the quinolones was associated with chromosomal mutations in thegyrA andparE genes. PCR analysis demonstrated that the genestetA,tetB,tetC,tetE,tetH,tetL, andtetM were not associated with the reduced susceptibility to oxytetracycline and the significance of this observation is discussed. PCR analysis also showed the presence ofsul2 in six isolates andfloR in one isolate. This study is the first report of these genes inF. psychrophilum.It was noted, however, that thefloR was detected in an isolate categorized as fully susceptible to florfenicol, but the reasons for its lack of expression in the phenotype was not investigated.It is argued that the Turkish trout industry, in common with those of many countries world-wide, is critically dependent on the continued susceptibility ofF. psychrophilumto florfenicol.

Otoliths of perch ( Perca fluviatilis ), pike ( Esox lucius ) and European whitefish ( Coregonus lavaretus ) caught at different locations in the northern Baltic Sea along the Finnish west coast and at some rivers and lakes were subjected to elemental analyses with particle induced X-ray emission and laser ablation inductively coupled plasma mass spectrometry. The strontium concentration in otoliths from whitefish (∼3300 μg/g) was 2–3 times higher than that of perch and pike (∼1400 μg/g), while within species the strontium concentration of otoliths from fish caught at different locations was in the same range. The strontium concentrations were lowest in fish from the lakes (∼450 μg/g). Whitefish otoliths contained more zinc (∼60 μg/g) than those of pike (∼30 μg/g), while the zinc concentration in perch otoliths were below the detection limit. No spatial intraspecies variations in zinc concentrations were observed. X-ray diffraction showed that the otoliths consisted of aragonite solely.

Flavobacterium psychrophilum isolated from rainbow trout Oncorhynchus mykiss suffering from bacterial cold-water disease (BCWD) can dissociate into 2 morphological colony types, rough (R) and smooth (S). However, the presence of the 2 morphotypes in disease outbreaks has not yet been investigated thoroughly. We examined the occurrence of R and S morphotypes in rainbow trout from BCWD outbreaks and in unfertilized eggs from a hatchery. The isolated colony types were characterized by pulsed field gel electrophoresis (PFGE), plasmid analysis, and oxolinic acid susceptibility testing. From most outbreaks, both morphotypes were isolated, although the S type only was isolated from the majority of individual fish. PFGE analysis showed both diverse and indistinguishable genetic patterns among the concurrent morphotypes. While PFGE patterns common to both fish and egg isolates were identified, this was not always the case. Resistant and sensitive isolates of both colony types were isolated from individual disease out- breaks. The plasmid pattern was partly associated with the colony type, showing identical or com- pletely different patterns for the R and S types isolated from the same outbreak. The study showed that within a BCWD outbreak, F. psychrophilum cells with different morphology, plasmid content, antibiotic susceptibility, and PFGE pattern can be isolated, suggesting that F. psychrophilum populations infecting rainbow trout in farm environments can be diverse and thus complicate the control of the disease.

International audience; Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

The adherence of Flavobacterium psychrophilum to surfaces of epithelial tissues has been inconclusively suggested as a mechanism, which enables the bacterium to invade the host. Hence, the present study aimed to examine the adherence of the cells of two colony phenotypes, smooth and rough, of F. psychrophilum to mucosal tissues of rainbow trout fry and to test the skin mucus as a nutrient for the growth of F. psychrophilum. Fish were immersed in water containing 106 CFU mL-1 F. psychrophilum for each colony phenotype. Mucosal tissue samples from fins, gills, skin and eyes, and swab samples from spleen and kidney were taken and inoculated onto TYES agar plates. Colony phenotypes of F. psychrophilum were identified and number of colonies counted. The results showed that cells of both phenotypes initially (0 h) adhered to all mucosal surfaces, but only the rough cells were still present on tissues 1 h post-immersion. Both phenotypes showed a tissue tropism with the fin tissue being the most adhered. Furthermore, skin mucus promoted the growth of both colony phenotypes. We suggest that the growth of F. psychrophilum cells in skin mucus apparently facilitates the bacterial adherence to mucosal surfaces, and the subsequent invasion into the host.

The cytotoxic activity of smooth and rough phenotypic cells of the fish pathogenic Gram-negative bacterium Flavobacterium psychrophilum to rainbow trout (Oncorhynchus mykiss) head kidney macrophages was investigated in vitro. The cytotoxicity to macrophages was significantly higher for rough cells compared with the smooth cells. The cytotoxic activity increased for both cell types with increasing temperature and the cells retained their cytotoxic nature after metabolical inactivation by heat, suggesting a cell-bound cytotoxic mechanism. The cytotoxicity was significantly reduced in both cell types after treatment with sodium (meta)periodate, indicating that the major bacterial structure involved in the cytotoxicity is of carbohydrate nature. Trypsin treatment further reduced the cytotoxicity in smooth cells, while sialic acid treatment reduced the cytotoxicity in rough cells, suggesting different lysing mechanisms for the two phenotypic variants. The results from the present study therefore suggest that the cytotoxic activity of F. psychrophilum to rainbow trout macrophages in vitro is stronger expressed in the rough phenotype and that it is opsonin-independent and initiated by binding of bacterial surface carbohydrates to lectins on the surface of the macrophages. How the lysis of the macrophages is executed is still unclear but it is suggested to function by different mechanisms in the smooth and the rough cells. The migration of rainbow trout macrophages toward smooth and rough cells of F. psychrophilum was further investigated. The results show that the macrophages were able to recognize both cell types, but the migration rate did not differ between the two phenotypes.

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.

From March 1994 to March 1998, breast cancer patients (an estimated relapse risk with 70% or more within five years with standard therapy) were randomised to treatment with tailored fluorouracil, epirubicin, and cyclophosphamide (FEC) therapy or FEC followed by marrow-supported high dose therapy in the Scandinavian Breast Group 9401 study. The aim of the present paper was to investigate differences in toxicity and eight health-related quality of life (HRQoL) variables (physical functioning, role functioning, emotional functioning, social functioning, cognitive functioning, fatigue, nausea-vomiting, and global quality of life) between women in the six dose steps used in the tailored and granulocyte colony stimulating factor supported FEC-arm at the assessment point 16 weeks after random assignment to treatment.The European Organization and Treatment of Cancer Quality of Life Questionnaire EORTC QLQ-C30 were mailed to the patients.A total of 157 (87%) in the tailored FEC-group responded to the questionnaire within the time frame 16 weeks after inclusion in the study. Overall, toxicity was low, reaching grade 1-2 also in the higher dose steps. There were no overall differences between the dose steps on any of the tested HRQoL variables. Patients at dose step 4 scored statistically significantly higher on physical functioning than patients at dose step 1 (p = 0.022) and compared to those at dose step 2 (p = 0.014). Patients at dose steps -2 and -1 (combined to one group) reported statistically significantly higher mean scores on cognitive functioning than patients at dose step 1 (p = 0.022).Patients who received higher doses, based on the tailored dosing strategy, did not seem to have worse HRQoL than those who had lower doses.

A Pseudomonas sp. isolate MSB1 efficiently inhibited the growth of Flavobacterium psychrophilum of different serotypes on agar medium. A significant difference in the inhibition was observed between isolates of the less virulent Fp(T) serotype compared to the Fd and Th serotypes. In broth coculture experiments, a low number of cells of MSB1 inhibited and outcompeted the F. psychrophilum cells. Also cell-free culture supernatant of MSB1 clearly repressed the growth of F. psychrophilum. A chromoazurol S assay suggested that MSB1 produced efficient siderophores, which most probably were responsible for the iron deficiency in the supernatant. The limited growth of F. psychrophilum in the supernatant was found to be partly because of the lack of available iron, but the results also indicated that some other mechanisms were probably involved in the observed inhibition. A potential use of MSB1 as a probiotic in rainbow trout aquaculture, especially in early life stages of the fish, is suggested, but future in vivo experiments needs to be carried out to verify this suggestion. This study also indicates a low iron acquisition efficiency of F. psychrophilum, compared to other examined bacterial fish pathogens.

Number of enteric redmouth disease (ERM) outbreaks, caused by the bacterial fish pathogen Yersinia ruckeri, has increased considerably in Finnish fish farms since 2004. To evaluate if the Y. ruckeri isolates from the recent severe Finnish outbreaks represent a dominant strain and if they differ from older isolates, 44 Finnish isolates from 1986 to 2007 were characterised by biochemical tests, serotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein analysis. The Finnish isolates were also compared with isolates from nearby geographical regions (Denmark and Sweden) and with the reference strain NCTC 10476. The results showed that most of the new isolates (2005–2007) from the Archipelago Sea (northern Baltic Sea) in Southwest Finland belong to biotype 2, which had not been previously reported from Finland. PFGE analysis indicated that the new biotype 2 isolates were very similar to isolates from Denmark (pulsotype 35) and that they most likely do not originate from the biotype 1 isolates obtained from fresh water hatcheries in Finland. Our conclusion is that the Y. ruckeri biotype 2 strain was introduced to Finnish fish farms in the Archipelago Sea from elsewhere.

Four 'smooth' and 4 'rough' colony phenotypes of the Gram-negative fish pathogen Flavobacterium psychrophilum isolated from rainbow trout Oncorhynchus mykiss were characterized using biochemical, physiological, molecular and virulence tests to better understand the pathogenesis of the bacterium. Biochemically, the 2 cell types did not react significantly differently. Physiologically, the 2 phenotypes had distinct characteristics, and, when grown in broth, the smooth cells were found to be autoagglutinating and able to switch into the non-agglutinating rough phenotype. The rough cells did not switch into the smooth phenotype under any growth conditions tested, indicating that the phase variation from the smooth to rough phenotype is irreversible or that the conditions for the reversible switch are still to be found. Smooth cells were hydrophobic and more adhesive compared to the hydrophilic rough cells, suggesting that the phase variation most probably involves one or several surface structures other than outer membrane proteins and lipopolysaccharides that were found to be similar in both types. Analysis of extracellular products produced by the 2 cell types indicated furthermore that a difference in enzymatic activities could exist. Both cell types were virulent for rainbow trout in an intramuscular challenge; thus, the distinct physiological characteristics of the phenotypes do not seem to be directly associated with virulence, when the body surface of the fish is disregarded. The results suggest that phase variation occurs in F. psychrophilum, but that the importance of the 2 phenotypes for the pathogenesis of the bacterium has still to be investigated.

Environmental estrogens are substances that imitate the effects of endogenous estrogens. Effluents from municipal wastewater treatment plants are known to contain substances with estrogenic activity including steroidal estrogens and xenoestrogens. In the current study, a combination of biological and chemical analysis was applied to determine the estrogenic activity in municipal wastewater effluents in Finland. The male three-spined stickleback (Gasterosteus aculeatus) hepatocyte assay with vitellogenin induction as an endpoint was used for the detection of estrogenic activity in solid phase extracts of wastewater effluents, and 17beta-estradiol (E2) as a positive control. The wastewater extracts and E2 were found to induce vitellogenin production. The extracts were also subjected to chromatographic fractionation and the collected fractions were assayed. The only active fraction was the one in which E2, estrone and ethynylestradiol were eluted. Its activity corresponded to the activity of the original wastewater extract. The LC-MS/MS analyses of the wastewater extracts showed that the concentration of estrone was about 65 ng L(-1), the concentration of E2 was less than 1 ng L(-1), while estriol and 17alpha-ethynylestradiol could not be detected. These findings showed that the activity of the wastewater extracts and the chromatographic fraction was much higher than the activity which could have been expected on the base of the chemical analysis. This strongly indicates that other compounds, possibly acting by additivity or synergism, are playing a major role in the induced vitellogenin production by the hepatocytes.

Based on randomized studies bone-marrow supported (BMS) high-dose chemotherapy (HDCT) is not superior to conventional CT as adjuvant treatment for high-risk breast cancer. To compare the cost-effectiveness of these treatments we examined the data of Finnish patients in the SBG9401 trial 1. Patients were randomized to receive either dose-escalated (de FEC) (group A, n =59) or FEC and HDCT+BMS (group B, n =70). They received adjuvant radiotherapy (RT) + tamoxifen. All direct health care costs of first line treatment at the oncology units were considered as well as productivity costs within the first 3 years of follow-up. Effectiveness was measured by the number of survival days during 5 years of follow-up. The mean direct health care costs were significantly higher in group B (25829 euro in group A vs. 36605 euro in group B, p0.001), mainly due to a higher number of hospital days. Half of the costs in group A was due to the use of filgrastim (15335 euro in A and 2969 euro in B, p0.001). The costs of RT were only 5% of total costs. There was no statistically significant difference between the groups in the number of survival days, but sensitivity analysis based on bootstrapping suggested that treatment A would be a less costly and more effective alternative in a great majority of cases.

Purpose Amplification of the HER-2/neu and topoisomerase IIα (TOP2A) genes has been linked to the effects of anthracyclines. Their role in predicting the outcome of anthracycline-based adjuvant chemotherapy for breast cancer patients has remained controversial. Patients and Methods The present substudy of the Scandinavian Breast Group trial 9401, in which an epirubicin-based regimen (nine courses of tailored and dose-escalated fluorouracil, epirubicin, and cyclophosphamide [FEC]) was compared with three or four courses of standard FEC followed by bone marrow–supported high-dose chemotherapy (cyclophosphamide, thiotepa, and carboplatin), included high-risk breast cancer patients (with eight or more positive axillary lymph nodes or at least five nodes with additional poor prognostic indicators). Amplification of HER-2/neu was determined retrospectively in paraffin-embedded tumor tissue sections by chromogenic in situ hybridization. TOP2A was tested only in HER-2/neu–amplified tumors. Results HER-2/neu amplification alone, which was present in 32.7% of the tumors, was a strong prognostic factor for short relapse-free (P = .0034) and overall survival (P = .0008) but showed no direct association with treatment assignment. TOP2A coamplification, which was present in 37% of HER-2/neu–amplified tumors, was associated with better relapse-free survival in patients treated with tailored and dose-escalated FEC (hazard ratio [HR] = 0.45; P = .049). A statistical multivariate Cox's regression analysis confirmed the predictive significance of TOP2A coamplification (HR = 0.30; P = .020) in HER-2/neu–amplified tumors. There was no such association in patients with HER-2/neu–amplified tumors without TOP2A gene amplification. Conclusion Coamplification of HER-2/neu and TOP2A may define a subgroup of high-risk breast cancer patients who benefit from individually tailored and dose-escalated adjuvant anthracyclines.

Invasive fungal infections (IFI) constitute a majorcomplication in allogeneic stem cell transplant(SCT) recipients, with an incidence of about 15%(1, 2). During the recent years, Aspergillus infec-tions have been predominant in this setting (1–4).In autologous stem cell transplant (ASCT)recipients, the risk of IFI has been considered tobe small when compared with allogeneic patients(5, 6). Several small single-centre series have shownan incidence of 1–2% or even less (7–11), althoughhigher figures have been observed especially inpatients receiving bone marrow support instead ofblood stem cells (12, 13). Recently, an incidence upto 5% for invasive aspergillosis (IA) in an autol-ogous setting has been reported from Seattle (14).The risk of Candida infections in autologous bonemarrow transplant recipients has been high in someearlier studies (15, 16).Several changes in autologous transplant pro-cedure have occurred during the last decade.These include the use of blood stem cells insteadof marrow cells, the use of granulocyte-colony-stimulating factor (G-CSF) after stem cell infu-sion, the use of manipulated stem cell grafts andfluconazole prophylaxis. In addition, improveddiagnosis of Aspergillus infections, includingJantunen E, Salonen J, Juvonen E, Koivunen E, Siitonen T, Lehtinen T,Kuittinen O, Leppa¨ S, Anttila V-J, Ita¨la¨ M, Wiklund T, Remes K,Nousiainen T. Invasive fungal infections in autologous stem celltransplant recipients: a nation-wide study of 1188 transplanted patients.Eur J Haematol 2004: 73: 174–178. Blackwell Munksgaard 2004.Abstract: Based on small single-centre series, the risk of invasive fungalinfections (IFI) has been considered small in autologous stem celltransplant (ASCT) recipients. Purpose: To analyse epidemiologicaland clinical features of (IFI) among ASCT recipients in Finland 1990–2001. Patients: During the study period, 1188 adult patients receivedhigh-dose therapy supported by ASCT in six centres. Altogether, 1112patients (94%) received blood progenitor cells. The graft was CD34+selected in 261 patients (22%). The major diagnostic groups werenon-Hodgkin’s lymphoma (n ¼ 417), multiple myeloma (n ¼ 395),breast cancer (n ¼ 132) and Hodgkin’s lymphoma (n ¼ 53).Results: Eighteen patients (1.5%) with IFI were identified. The inci-dence of proven or probable invasive aspergillosis was 0.8%, followedby candidaemia with an incidence of 0.3%. The median time to thediagnosis of IFI was 35 d (6–162) from the progenitor cell infusion. Infourteen patients (78%) IFI was diagnosed during lifetime and they weretreated with antifungal therapy for a median of 50 d. Nine patients(64%) were cured. Conclusions: IFI appears to be a rare event afterASCT and Aspergillus infections seem to be predominant. These epi-demiological features have an impact in planning prophylactic andempirical antifungal strategies in ASCT recipients.E. Jantunen

Limited experience is available on the feasibility and efficacy of high-dose therapy (HDT) supported by autologous stem cell transplantation (ASCT) in patients with peripheral T-cell lymphoma (PTCL). Therefore, a nation-wide survey was conducted in adult patients transplanted for PTCL in Finland during 1990-2001. After histopathology review, 37 patients were identified. The median age was 46 years (16-68) at the time of ASCT. Histology included PTCL not otherwise specified in 14 patients, anaplastic large cell lymphoma (ALCL) in 14 patients, and other in nine patients. Disease status at the time of ASCT was CR/PR1 in 18 patients; CR/PR2 in 14 patients, and other in five patients. HDT consisted of either BEAC (N=22) or BEAM (N=15), supported by blood stem cells in 34 patients (92%). Early transplant-related mortality was 11%. With a median follow-up of 24 months from HDT, 16 patients (43%) have relapsed or progressed. The estimated 5-year overall survival (OS) was 54%. Patients with ALCL had superior OS when compared with other subtypes (85 vs 35%, P=0.007). OS at 5 years was 63% in patients transplanted in CR/PR1 vs 45% in those transplanted in other disease status (P=NS). Prospective studies are needed to define the role of ASCT in this lymphoma type.

From 1990 to 1997, 113 eligible patients with classical osteosarcoma received neo-adjuvant chemotherapy consisting of high-dose methotrexate, cisplatin and doxorubicin. Good histological responders continued to receive the same therapy postoperatively, while poor responders received salvage therapy with an etoposide/ifosfamide combination. With a median follow-up of 83 months, the projected metastasis-free and overall survival rates at 5 years are 63 and 74%, respectively. Independent favourable prognostic factors for outcome were tumour volume 4.5 μM and female gender. The etoposide/ifosfamide replacement combination did not improve outcome in the poor histological responders. In conclusion, this intensive multi-agent chemotherapy results in >70% of patients with classical osteosarcoma surviving for 5 years. The data obtained from this non-randomised study do not support discontinuation and exchange of all drugs used preoperatively in histological poor responders. As observed in previous Scandinavian osteosarcoma studies, female gender appears to be a strong predictor of a favourable outcome.

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome and cold water disease in salmonids, causes serious disease outbreaks in fish farms worldwide. The aim of the present study was to examine the survival capacity of F. psychrophilum in laboratory microcosms containing sterilised water under different environmental conditions and to examine the virulence of starving F. psychrophilum cells. The results showed that F. psychrophilum survived for very long time in sterilised fresh water at 15 degrees C and the cells were still culturable after starvation for 300 days. A high salinity of the water (30 per thousand) drastically reduced the number of culturable cells below detection limit after incubation for 1 day. A water salinity of approximately 6 per thousand initially reduced the number of culturable cells below the detection limit, but cells were again recovered on agar plates at the end of the experiment. The presence of sediment containing nutrients in the experimental water microcosms increased the survival of F. psychrophilum. The challenge experiments indicated that the virulence of starving F. psychrophilum is maintained for at least seven days after the transfer of the bacterial cells to fresh water.

ContextSecond cancer is the leading cause of death in long-term survivors of Hodgkin disease (HD), with exceptionally high risks of breast cancer among women treated at a young age. Quantitative associations between radiotherapy dose delivered to the breast and administered chemotherapy have not been reported to date in large series, nor has the influence of ovarian exposures on subsequent risk.ObjectiveTo quantify the long-term risk of breast cancer associated with use of radiotherapy and chemotherapy to treat young women with HD.Design, Setting, and SubjectsMatched case-control study of breast cancer within a cohort of 3817 female 1-year survivors of HD diagnosed at age 30 years or younger, between January 1, 1965, and December 31, 1994, and within 6 population-based cancer registries. The study was conducted March 1, 1996, through September 30, 1998.Main Outcome MeasuresRelative risk (RR) of breast cancer associated with radiation dose delivered to site of breast cancer or to ovaries and with cumulative dose of alkylating agents.ResultsBreast cancer occurred in 105 patients with HD who were matched to 266 patients with HD but without breast cancer. A radiation dose of 4 Gy or more delivered to the breast was associated with a 3.2-fold (95% confidence interval [CI], 1.4-8.2) increased risk, compared with the risk in patients who received lower doses and no alkylating agents. Risk increased to 8-fold (95% CI, 2.6-26.4) with a dose of more than 40 Gy (P