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Flavobacterium psychrophilum. Typical appearance of (A) a smooth and (B) a rough colony type, grown on tryptone yeast extract salts (TYES) agar. (C) In TYES broth the smooth colony type cells autoagglutinated at the bottom of the tube (left), while the rough colony type cells showed a non-agglutinating and uniform growth mode (right)
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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 signifi...
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Context 1
... isolates and growth conditions. The 4 Flavobacterium psychrophilum isolates (P13-4/96, P6- 1/07, P6-3/07 and P6-8/07) used in the present study were all previously isolated in 1996 and 2007 from spleen or kidney of diseased farmed rainbow trout Oncorhynchus mykiss at our laboratory. All isolates contained smooth (S) (Fig. 1A) and rough (R) (Fig. 1B) colony phenotypic variants that were separated for the study and thereafter named after the corresponding colony type as P13-4S/96, P13-4R/96, P6-1S/07, P6- 1R/07, P6-3S/07, P6-3R/07, P6-8S/07 and P6-8R/07. All variants were identified as F. psychrophilum by poly- merase chain reaction (PCR) (Wiklund et al. ...
Context 2
... isolates and growth conditions. The 4 Flavobacterium psychrophilum isolates (P13-4/96, P6- 1/07, P6-3/07 and P6-8/07) used in the present study were all previously isolated in 1996 and 2007 from spleen or kidney of diseased farmed rainbow trout Oncorhynchus mykiss at our laboratory. All isolates contained smooth (S) (Fig. 1A) and rough (R) (Fig. 1B) colony phenotypic variants that were separated for the study and thereafter named after the corresponding colony type as P13-4S/96, P13-4R/96, P6-1S/07, P6- 1R/07, P6-3S/07, P6-3R/07, P6-8S/07 and P6-8R/07. All variants were identified as F. psychrophilum by poly- merase chain reaction (PCR) (Wiklund et al. 2000). The F. psychrophilum ...
Context 3
... TYES broth, the smooth variants demonstrated a distinct growth mode, whereby the cells auto- agglutinated and precipitated in clusters at the bottom of the tube or adhered to the tube walls. The rough cells, on the other hand, had a uniform growth mode, resulting in a turbid solution (Fig. 1C). In TYES broth, the rough variants did not change their turbid appear- ance over time, and the colony type was constantly identified as rough when dilutions of the broth were in- oculated on TYES agar (data not shown). However, the growth of the smooth variants shifted from a clear broth with precipitated cell clusters to a more turbid ...
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The possibility of seroIogical differentiation between isolates of Flavobacterium psychrophilum was analyzed by ELISA and slide agglutination. Twenty-five Danish isolates and 20 isolates from other European countries were studied using polyclonal rabbit antisera and whole-cell preparations. Unabsorbed as well as reciprocally absorbed antisera and p...
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Background
Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067...
Citations
... This assumption is supported by experiments in which highly virulent isolates appear to have strong adhesive activity, whereas the adhesion of low-virulent isolates is weaker [26], and three-dimensional structure may be absent [13]. However, some studies have noted that adherence ability is strongly related to colony morphological type [35][36][37], regardless of virulence [35,37]. Thus, adhesion and biofilm formation are presumably not crucial determinants of strain virulence. ...
... This assumption is supported by experiments in which highly virulent isolates appear to have strong adhesive activity, whereas the adhesion of low-virulent isolates is weaker [26], and three-dimensional structure may be absent [13]. However, some studies have noted that adherence ability is strongly related to colony morphological type [35][36][37], regardless of virulence [35,37]. Thus, adhesion and biofilm formation are presumably not crucial determinants of strain virulence. ...
... isolates is weaker [26], and three-dimensional structure may be absent [13]. However, some studies have noted that adherence ability is strongly related to colony morphological type [35][36][37], regardless of virulence [35,37]. Thus, adhesion and biofilm formation are presumably not crucial determinants of strain virulence. ...
Flavobacterium psychrophilum is currently one of the most important pathogens in aquaculture worldwide, causing high losses to farmed salmonids particularly during early growth stages with significant economic impact. Despite previous attempts, no effective vaccine has been developed, and protection against introduction into farms is difficult due to the ubiquitous occurrence of the pathogen. A better understanding of the mechanism of disease development is essential for targeted therapeutic and preventive measures in farms. Unfortunately, the pathogenesis of diseases caused by F. psychrophilum has not been elucidated yet. Previously, several putative virulence factors have been identified. Some appear to be essential for disease development, while others are probably dispensable. The importance of some factors has not yet been explored. This review focuses on the supposed virulence factors of F. psychrophilum and the current knowledge about their importance in the pathogenesis of the disease.
... CC-ST10 isolates from BCWD outbreaks in rainbow trout have previously been associated with a higher ability to adhere to inert and mucus coated surfaces compared to genetically distinct F. psychrophilum strains isolated from other sources (Sundell and Wiklund, 2015). Our results confirmed previous observations that smooth colony phenotypes are superior to rough in adhering to inert surfaces (Högfors-Rönnholm et al., 2015) but equally virulent (Figure 2; R 2 = 0.04, P = 0.13) when injected into rainbow trout (Högfors-Rönnholm and Wiklund, 2010b). Similarly, virulence in F. psychrophilum could not be predicted by the hemolytic activity in the microplate assay, but here, contrary to previous findings by Högfors-Rönnholm and Wiklund (2010a), we did not detect any significant differences (R 2 = 0.01, P = 0.59) in the activity between rough and smooth isolates. ...
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 × 103 to 3.1 × 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 < 105 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.
... Flavobacterium psychrophilum, a ubiquitous fish pathogen that can be found in many temperate freshwater farming environments, still remains a bottleneck for rainbow trout Oncorhynchus mykiss hatcheries by causing significant mortalities. F. psychrophilum occurs in two different colony phenotypes, smooth and rough (Högfors-Rönnholm and Wiklund, 2010) which have different adherence capacity to inert and living surfaces (Papadopoulou et al., 2015;Papadopoulou et al., 2017). Both colony phenotypes have frequently been isolated from the same fish individuals within a disease outbreak (Sundell et al., 2013). ...
The fish pathogen Flavobacterium psychrophilum has been shown to occur in two colony phenotypes, rough and smooth. The aim of this study was to examine the effect of environmental and nutritional factors on the biofilm formation of both phenotypes under in vitro static conditions. The present study revealed that smooth cells displayed higher abilities of biofilm formation compared to rough cells in nutrient-rich media (TYES broth and fish feed) under static conditions, while biofilms formed by cells of both phenotypes were mainly Ca²⁺ dependent than Mg²⁺. Neither of the colony phenotypes was able to form biofilms in the low-nutrient medium (lake water) within 5 days.
... Flavobacterium columnare is generally found in a planktonic (free living) state, surviving where it can for long periods of time in the absence of suitable nourishment (Arias et al. 2012). Flavobacterium sp. also possesses an ability to form biofilms both on inanimate and biological surfaces (Hogfors-Ronnholm and Wiklund 2010;Cai, De La Fuente and Arias 2013). The advantages of adopting the biofilm life stage are not fully understood but we and others have demonstrated that biofilms increase the overall virulence of Flavobacterium sp. ...
Columnaris disease, caused by Flavobacterium columnare, severely impacts the production of freshwater finfish species. Therefore, efforts to better understand the biological processes of F. columnare, including the formation of biofilms and their contribution to disease, are ongoing. In this study, we incubated F. columnare cultures with channel catfish mucus and used high-throughput RNA sequencing to evaluate global changes in gene expression. Our data shows that mucus activates in vitro biofilm formation. The analysis of F. columnare transcriptomes after the addition of mucus revealed significant differentially expressed genes (DEGs) between the planktonic and biofilm states. DEGs common among all biofilms were enriched for gene ontology groups including signal transduction, ligand binding and cellular homeostasis and are likely necessary for biofilm formation. Iron acquisition systems included TonB dependent receptor and ferroxidase genes were expressed among all biofilms, while siderophore synthesis genes were only expressed in mucus-stimulated biofilms. The current analysis of F. columnare transcriptomes adds valuable information about the basic biological processes that occur during the planktonic and biofilm states. This work serves as a basis for future studies on understanding how biofilms are established and how they contribute to disease progression.
... Moderate to severe lesions in internal organs such as focal necrosis in spleen and kidney often occur in diseased rainbow trout (Barnes & Brown 2011). Two distinct colony phenotypes of F. psychrophilum, smooth and rough, have previously been reported by H€ ogfors-R€ onnholm & Wiklund (2010a). Colonies of both phenotypes have been isolated from diseased rainbow trout, and both rough and smooth cell types were able to cause mortality after intramuscular injection in rainbow trout (H€ ogfors-R€ onnholm & Wiklund 2010a). ...
... Two distinct colony phenotypes of F. psychrophilum, smooth and rough, have previously been reported by H€ ogfors-R€ onnholm & Wiklund (2010a). Colonies of both phenotypes have been isolated from diseased rainbow trout, and both rough and smooth cell types were able to cause mortality after intramuscular injection in rainbow trout (H€ ogfors-R€ onnholm & Wiklund 2010a). Despite extensive studies on genotype and phenotype variation of F. psychrophilum (H€ ogfors-R€ onnholm 2014;Sundell 2015), the role of the phase variation in the pathogenesis has not been investigated yet. ...
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 10(6 ) 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.
... Epidemic STs were genetically associated with the globally spread CC-ST2 (Fig. 1) and were considered part of the same lineage whereas sporadic STs consisted of singletons and minor CCs genetically distinct from CC-ST2. The colony morphology of F. psychrophilum is either rough or smooth (Rönnholm and Wiklund, 2010) when grown on tryptone yeast extract salts (TYES) (Holt et al., 1993) agar. The rough and smooth F. psychrophilum morphotypes have been shown to express different phenotypic characteristics in vitro (Högfors-Rönnholm, 2014). ...
... Some of these virulence mechanisms are, for example, secreted proteases important for host tissue breakdown (Bertolini et al., 1994) and nutrient acquirement (Pérez-Pascual et al., 2011), hemolysins for cell destruction and nutrient acquisition (Högfors-Rönnholm and Wiklund, 2010b), adhesins for attachment to host tissues (Kondo et al., 2002;Nematollahi et al., 2003a) and cells (Møller et al., 2003;Wiklund and Dalsgaard, 2003;Högfors-Rönnholm and Wiklund, 2010b) and proteins involved in biofilm formation (Álvarez et al., 2006). Since F. psychrophilum expresses both antigenic Lorenzen and Olesen, 1997) and phase variation (Högfors-Rönnholm and Wiklund, 2010a), it is challenging to determine the overall virulence mechanisms for this pathogen as different types probably express diverse virulence mechanisms. It has been shown that even though all F. psychrophilum isolates share some antigens, they can be divided into three Lorenzen and Olesen, 1997) or seven (Mata et al., 2002) distinct serotypes. ...
In recent years, an increasing number of Flavobacteriaceae species have been associated with infections in fish. Still, diseases caused by Flavobacterium branchiophilum (bacterial gill disease), F. columnare (columnaris disease), F. psychrophilum (bacterial cold-water disease), and Tenacibaculum maritimum (tenacibaculosis) have been more extensively studied due to their significant economic impact on the aquaculture industry. So far, commercial vaccines have been made available only against columnaris disease, while protection against bacterial cold-water disease has relied on autogenous vaccines. In this chapter, the main focus is on immunization against diseases caused by Flavobacterium branchiophilum, F. columnare, F. psychrophilum, and Tenacibaculum maritimum. These microorganisms are infecting a large number of fish species worldwide, causing significant problems in both marine and freshwater aquaculture.
... F. psychrophilum from rainbow trout included isolates from eggs (n ϭ 4), ovarian fluid (n ϭ 10), and milt (n ϭ 12). Environmental isolates from water samples (n ϭ 11) from fish farm environments, as well as three rough laboratory mutant strains prepared from isolated smooth colony types by repeated passages in TYES broth (34), were included in the collection. ...
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD) affecting a variety of freshwater-reared salmonid species. A large scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway and Sweden. Multilocus sequence typing (MLST) of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution towards mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.
... Flavobacterium psychrophilum is globally distributed with an apparently low level of diversity and high recombination rate based on nucleotide polymorphism among 11 protein-coding loci (Nicolas et al., 2008), suggesting high species homogeneity on a global scale. Despite its global distribution and severe pathogenic implications, little is still known about the genomic diversity, and the factors controlling the genetic recombination and virulence of F. psychrophilum (H€ ogfors-R€ onnholm & Wiklund, 2010). ...
Flavobacterium psychrophilum is currently one of the most devastating fish pathogens worldwide causing considerable economic losses in salmonid aquaculture. Recently, attention has been drawn to the use of phages for controlling F. pyschrophilum and phages infecting the pathogen have been isolated. Here we present the genome sequence of F. psychrophilum bacteriophage 6H and its distribution as prophage in F. psychrophilum isolates. The DNA sequence revealed a genome of 46978 bp containing 63 predicted ORFs, of which 13 % was assigned a putative function, including an integrase. Sequence analysis showed >80% amino acid similarity to a specific region found in the virulent F. psychrophilum strain JIP02/86 (ATCC 49511), suggesting that a prophage similar to phage 6H was present in this strain. Screening for a collection of 49 F. psychrophilum strains isolated in Chile, Denmark and USA for the presence of four phage 6H genes (integrase, tail tape protein and two hypothetical proteins) by PCR showed the presence of these prophage genes in 80% of the isolates. In conclusion, we hypothesize that bacteriophage 6H belongs to an abundant group of temperate phages which has lysogenized a large fraction of the global F. psychrophilum community. This article is protected by copyright. All rights reserved.
... F. psychrophilum has previously been reported to stimulate [4], survive and multiply in [5], adhere to [6] and be cytotoxic to [7] rainbow trout (Oncorhynchus mykiss) phagocytes. In these studies however, the phase varying nature of F. psychrophilum, expressed as smooth and rough phenotypic variants [8], was not taken into consideration. In our previous study, we demonstrated different hemolytic activity in smooth and rough phenotypes of F. psychrophilum isolated from rainbow trout. ...
... The phenotype of these F. psychrophilum cells was however not noted [7]. Both smooth and rough cells have been reported to cause disease in rainbow trout [8]. In the future it needs to be investigated whether the less cytotoxic smooth F. psychrophilum cells are able to survive and multiply better in macrophages compared with the Fig. 1. ...
... The rough surface, a consequence of the reduction of a glycopeptidolipid in the cell wall associated with motility and biofilm formation in the smooth variant, was suggested to be the reason for this. The rough variant was also reported to be more virulent than the smooth variant in mice infection models [19] which does not, however, seem to be the case for the smooth and rough variants of F. psychrophilum as both are virulent for rainbow trout when injected [8]. ...
