Phenotypic and genotypic characterization of Flavobacterium psychrophilum from Finnish fish farms

Finnish isolates ( n =37) of Flavobacterium psychrophilum isolated from farmed salmonids were studied using phenotypic and genotypic characteristics. The characteristics of isolates were compared with the characteristics of Swedish and Estonian F. psychrophilum isolates and the type strain, F. psychrophilum NCIMB 1947^T. Multiple isolates from eight disease outbreaks were examined to determine differences between isolates from a single outbreak. The F. psychrophilum isolates represented a biochemically homogeneous group. However, some minor differences in biochemical and physiological characteristics were observed. Seven different antigenic patterns among Finnish isolates were detected and the results suggest a new serotype of F. psychrophilum . Using Cla Ι, Hae ΙΙΙ and Pvu ΙΙ restriction enzymes in ribotyping analyses 13 different genotypes were demonstrated and a possible relationship between serotype Fd and genotype F1 was determined. There were no significant differences between the isolates from Finland, Estonia, Sweden and the type strain of F. psychrophilum .     

Yersinia ruckeri infections in salmonid fish

Yersinia ruckeri is the causative agent of yersiniosis or enteric redmouth disease leading to significant economic losses in salmonid aquaculture worldwide. Infection may result in a septicaemic condition with haemorrhages on the body surface and in the internal organs. Despite the significance of the disease, very little information is available on the pathogenesis, hampering the development of preventive measures to efficiently combat this bacterial agent. This review discusses the agent and the disease it causes. The possibility of the presence of similar virulence markers and/or pathogenic mechanisms between the Yersinia species which elicit disease in humans and Y. ruckeri is also examined.     

Growth of Flavobacterium psychrophilum in fish serum correlates with pathogenicity

Flavobacterium psychrophilum isolates, obtained from ayu, Plecoglossus altivelis, three species of salmonids and two species of cyprinids in Japan, were used in this study. Bacteria were inoculated to serum prepared from ayu or red spotted masu trout (RSMT), Oncorhynchus masou ishikawae, and incubated at 18 °C for 24 h. All isolates (n = 19) from ayu grew well with a 9- to 116-fold increase of CFU in ayu serum, while CFU decreased markedly in RSMT serum. In contrast, isolates (n = 17) from fish species other than ayu exhibited no growth in ayu serum, but some isolates from salmonids survived or grew (1.2-23.5 fold increase of CFU) in RSMT serum. The isolates that could not survive or grow in ayu and RSMT sera grew well in both heat-inactivated sera of ayu and RSMT. Experimental infection by intraperitoneal injection showed that ayu isolates examined were all pathogenic to ayu but not to RSMT, while none of the isolates from salmonids and cyprinids were pathogenic to ayu but some showed pathogenicity to RSMT. These results indicate that the in vitro growth ability of F. psychrophilum isolates in fish serum correlates well with their pathogenicity to fish, particularly in ayu.     

Validation of diagnostic assays to screen broodstock for Flavobacterium psychrophilum infections

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

Molecular typing by pulsed-field gel electrophoresis of Flavobacterium psychrophilum isolates derived from Japanese fish

Sixty-four isolates of Flavobacterium psychrophilum from ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel), and other fish (n=16) in Japan and the type strain (NCIMB 1947(T)) were typed using pulsed-field gel electrophoresis (PFGE) with endonuclease BlnI and XhoI. These isolates were classified into 20 clusters and 42 genotypes by PFGE analysis. The most predominant cluster of isolates from ayu was cluster XII (n=20), followed by clusters XVII, XVI, XX, XI, IX, X, XIII and XV; the remaining 17 isolates from other fish were divided into clusters I, II, III, IV, V, VI, VII, VIII, XIV, XVIII and XIX. The PFGE genotype of isolates from ayu clearly differed from those of other fish. The isolates from ayu in Gunma Prefecture belonged to clusters XII, XVI, XVII and XX, and the strains of three of these clusters (XII, XVII and XX) were isolated from ayu in 15 of 19 prefectures. PFGE typing enabled more accurate classification of isolates into clusters than previously achieved by analysing the restriction fragment length polymorphism of PCR products. These results suggest that F. psychrophilum isolated from ayu and other fish are genetically different and strains with several PFGE types have spread within Japan.     

Comparative proteomic analysis of virulent and rifampicin-attenuated Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study, we compared a wild‐type strain (CSF 259‐93) with a rifampicin‐resistant strain and virulence‐attenuated strain of F. psychrophilum (CSF 259‐93B.17). The attenuated strain harboured a mutation in the rpoB gene consistent with resistance to rifampicin. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry demonstrated an altered proteome with eight proteins characteristic for the parent strain and six that were unique to the attenuated strain. Immunoblotting with a diagnostic monoclonal antibody (FL‐43) identified a putative antigen (FP1493) that was subsequently cloned, expressed as a recombinant protein and confirmed as recognized by FL‐43. 2D‐PAGE, immunoblotting with rainbow trout, Oncorhynchus mykiss (Walbaum), convalescent antisera and mass spectrometry of bacterial whole‐cell lysates revealed several uniquely expressed immunoreactive proteins including FP1493. An FP1493 recombinant subunit vaccine was tested, but did not provide protection against challenge with the CSF259‐93 strain. While the exact mechanism responsible for altered protein synthesis and attenuation of CSF 259‐93B.17 is still unknown, the differentially expressed immunoreactive proteins are a valuable resource to develop subunit vaccines and to identify proteins that are potentially involved in disease.     

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

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

Iron acquisition mechanisms of Flavobacterium psychrophilum

Forty strains of Flavobacterium psychrophilum were tested for the production of siderophores using the universal Chrome Azurol S (CAS) assay. The majority of the strains (85%) were CAS positive (CAS+) and some (15%) were CAS negative (CAS-). The cryptic plasmid pCP1 was carried by all positive strains and was lacking from negative strains. While a weak catechol reaction was detectable in CAS+ culture supernatants, the CAS reaction was, to some extent, heat sensitive, questioning whether the positive reaction was caused only by siderophores. The ability to grow in vitro under iron-restricted conditions did not correlate with the CAS reactivity, as growth of both CAS+ and CAS- strains was similarly impaired under iron restriction induced by 2,2 dipyridyl. Suppressed growth under these conditions was restored by addition of FeCl3, haemoglobin and transferrin for both CAS+ and CAS- strains.     

Inhibitory activity of Pseudomonas sp. on Flavobacterium psychrophilum, in vitro

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.     

Identification of immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD), and this pathogen has large economic impacts on salmonid aquaculture worldwide. Previously, it was demonstrated that high levels of protection against F. psychrophilum challenge were conferred to rainbow trout, Oncorhynchus mykiss (Walbaum), by immunization with distinct molecular mass fractions of the bacterium, and specific antibodies were correlated with protection. In this study, an immunoproteomic analysis of F. psychrophilum was performed using two-dimensional polyacrylamide gel electrophoresis and Western blotting with serum from fish immunized with high- and mid-molecular mass fractions of the bacterium. Mass spectrometry was used to determine the protein identity, and 15 immunogenic proteins were positively identified following Mascot searches of the F. psychrophilum genome. Based on known function and immunogenicity of homologous proteins in other bacterial pathogens, antibodies specific for several of the identified proteins may be important for protective immunity from CWD. These include outer membrane protein OmpA (P60), trigger factor, ClpB, elongation factor G, gliding motility protein GldN and a conserved hypothetical protein. This work increases the understanding of the protective humoral immune response of rainbow trout against these distinct molecular mass fractions of F. psychrophilum and provides new potential targets for recombinant protein vaccine development.     

Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum

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.     

Adhesion of smooth and rough phenotypes of Flavobacterium psychrophilum to polystyrene surfaces

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.     

PCR-RFLP genotypes associated with quinolone resistance in isolates of Flavobacterium psychrophilum

A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.     

Serological diversity in Flavobacterium psychrophilum: A critical update using isolates retrieved from Chilean salmon farms

Chile is currently the second largest producer of farmed salmon worldwide, but Flavobacterium psychrophilum, as one of the most detrimental pathogens, is responsible for major losses during the freshwater culturing step in salmonid fish farms. An antigenic study conducted 10 years ago reported four serological groups using 20 F. psychrophilum Chilean strains. To reduce disease outbreaks and to develop vaccine candidates, antigenic knowledge needs to be regularly updated using a significant number of additional recent F. psychrophilum isolates. The present study aimed at investigating the serological diversity of 118 F. psychrophilum isolates collected between 2006 and 2018 from farmed Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch). The current study supports an expansion of the known antigenic groups in Chile from 4 to 14. However, the use of the slide-agglutination technique for serotyping is costly, is labour-intensive and requires significant technical expertise. Addressing these points, the mPCR-based procedure was a very useful tool for serotyping the collected Chilean F. psychrophilum isolates. This technique revealed the presence of diverse mPCR serotypes (i.e. types 0, 1, 2 and 4). Therefore, mPCR should be employed to select the bacterial strain(s) for vaccine development and to conduct follow-up, selective breeding or epidemiological surveillance in Chilean fish farms. Given the presented findings, changes to Chilean fish-farming practices are vital for ensuring the continued productivity and well-being of farmed salmonids.     

In vivo study of phagocytosis, intracellular survival and multiplication of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), spleen phagocytes

The intracellular behaviour of a Flavobacterium psychrophilum strain, ingested by spleen phagocytes of rainbow trout, Oncorhynchus mykiss , of different ages, was assessed in vivo . Three groups of rainbow trout weighing 1 g (aged 10 weeks), 25 g (aged 20 weeks) and 300 g (aged 15 months), respectively, were injected intraperitoneally with 1 × 10⁶ cfu of a F. psychrophilum strain. It was found that only fry, aged 10 weeks, displayed clinical signs and suffered mortality. Bacteriological colony plating of different organs demonstrated that the spleen and to a lesser extent the kidney of only the fry were affected. The number of colony forming units per gram of spleen tissue increased with time. No bacteria were found in the trout aged 5 months and older. Light microscopical examination and epifluorescence microscopy revealed that the fry spleen phagocytes contained an increasing number of viable intracellular F. psychrophilum bacteria over time. Again, no bacteria were encountered in the phagocytes collected from older fish.     

Pathology and immunohistochemistry in three species of salmonids after experimental infection with Flavobacterium psychrophilum

Rainbow trout, Oncorhynchus mykiss (Walbaum), sea trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., were experimentally infected with Flavobacterium psychrophilum in order to evaluate any species differences in susceptibility to the bacterium. Furthermore, differences in pathological changes and distribution of the bacteria in internal organs were studied. The bacteria were injected intraperitoneally in two doses, high dose (Hd) 1 x 10(7) colony forming units (CFU) fish(-1) and low dose (Ld) 1 x 10(6) CFU fish(-1). The mortalities in the Ld groups varied between 0 and 7.5% and in the Hd groups between 55-70%. No significant differences in mortality between the species were recorded. Clinical signs and pathological findings were similar in the three species and in accordance with those of rainbow trout fry syndrome. Rainbow trout showed more pronounced lesions in the spleen compared with the other species. Necrosis of renal tubular epithelium and haematopoietic tissue was most prominent in rainbow trout and Atlantic salmon. Intracellular eosinophilic droplets in the kidney tubular epithelium were a prominent finding in rainbow trout and sea trout surviving the infection. The distribution of the bacteria in internal organs was similar in the three species, as studied with immunohistochemistry.