Tom Wiklund

Survival and virulence of Flavobacterium psychrophilum in water microcosms

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.

Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms.

Rainbow trout fry syndrome and cold-water disease are serious diseases in farmed salmonid fish. In the present study, three methods were compared, for the detection of the causative pathogen, Flavobacterium psychrophilum in water. The methods included traditional agar plate cultivation on tryptone yeast extract salts (TYES) agar, immunofluorescence antibody technique (IFAT) and nested PCR. The three methods were subsequently used for the detection of F. psychrophilum from fish farm environments. The nested PCR was the most sensitive method used for a detection of F. psychrophilum. As low as 3 CFU estimated by agar plate cultivation or 41 cells estimated by IFAT of F. psychrophilum per ml of non-sterile well water were needed for a detection using the nested PCR method. The obtained detection limits for the agar plate cultivation and the IFAT was 32 CFU/ml and 410 cells/ml, respectively. Using IFAT and nested PCR F. psychrophilum was detected most frequently in water samples from fish farms, but the pathogen was isolated from only a few samples using agar plate cultivation. In the present study IFAT and nested PCR proved to be rapid, specific and sensitive methods compared to traditional agar plate cultivation for the detection of F. psychrophilum from environmental samples. It is suggested that IFAT and nested PCR provide effective tools for the examination of F. psychrophilum in the environment.

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.

Estrogenic and androgenic effects of municipal wastewater effluent on reproductive endpoint biomarkers in three-spined stickleback (gasterosteus aculeatus)

Municipal wastewater treatment plants have been associated with the release of endocrine-disrupting chemicals, which consequently lead to alterations of reproductive function in aquatic organisms. The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers for assessment of both estrogen (vitellogenin) and androgen (spiggin) activity, which makes this species very valuable in the research of endocrine disruption. The estrogenic and androgenic biomarkers were used for evaluating exposure effects of municipal wastewater effluent. We evaluated the effects of 17alpha-ethinylestradiol (EE2), 17alpha-methyltestosterone (MT), and wastewater effluents on induction of vitellogenin and spiggin production, gonadosomatic index, hepatosomatic index, nephrosomatic index, plasma steroid levels, and histopathology. Adult female and male sticklebacks were exposed to 20 ng/L of EE2, 10 microg/L of MT, and wastewater effluent (10, 50, and 80% of original concentration) in a flow-through system for an exposure of one week and an extended exposure of four weeks. Chemical analyses of the steroids were done for verification of exposure concentrations and presence in the used wastewater. Our results show that municipal wastewater effluent exerts estrogenic action on three-spined stickleback as observed by elevated vitellogenin levels in exposed fish, corresponding to the effect seen in fish exposed to EE2. Furthermore, wastewater and EE2 exerted similar histopathological effects on testis of exposed fish. Although domestic effluent is suspected to have a high content of natural androgens, no obvious androgenic effect of wastewater was observed in the present study.

Bacterial growth in humic waters exposed to UV-radiation and simulated sunlight

Sterile filtered (0.45 μm) humic lake water was exposed to simulated sunlight (300–800 nm) or UV-radiation (254 nm) for various periods of times and the dissolved organic carbon content, absorbance at 254 and 460 nm and pH were recorded. The irradiated water was inoculated with a natural bacterial assemblage and the number of viable bacteria was estimated 3 and 5 days after the inoculation using the plate count technique. The number of viable bacteria increased with the irradiation time indicating that the chemical changes of the humus macromolecules observed during irradiation resulted in an increased availability of the dissolved organic material as bacterial substrate. No inhibitory effect on the bacterial growth, due to the presence of toxic organic reaction products, was observed.

Multilocus Sequence Typing Identifies Epidemic Clones of Flavobacterium psychrophilum in Nordic Countries

ABSTRACT Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects 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 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 toward 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.

Immersion exposure of rainbow trout (Oncorhynchus mykiss) fry to wildtype Flavobacterium psychrophilum induces no mortality, but protects against later intraperitoneal challenge

Flavobacterium psychrophilum, the causative agent of RTFS or rainbow trout fry syndrome, causes high mortality among hatchery reared rainbow trout (Oncorhynchus mykiss) fry in Europe and the USA. Despite several attempts, no efficient vaccines have yet been developed, the main obstacle being that the fry have to be vaccinated very early, i.e. around 0.2-0.5 g, where RTFS usually starts to give problems in the fish farms. Consequently, only oral or bath vaccines are relevant. Immersion of fry in inactivated or attenuated bacteria has resulted in RPS values of less than 50%. However, the results are biased by the fact that the fish have been challenged by intraperitoneal (ip) or subcutaneous (sc) injection against which an immersion/oral vaccine may not protect. Therefore, the present study was undertaken in order to investigate whether the presumably most potent immersion immunization, i.e. bathing in high titres of non-attenuated isolates of F. psychrophilum, was able to induce immunity to a subsequent ip challenge. Immersion in live bacteria for 30 or 50 min caused no mortality and protected a major fraction of the fry against challenges 26 and 47 days later with RPS values of 88.2 and 60.3%, respectively. Increased specific antibody titres suggested that adaptive immune mechanisms were involved in the protection.

Detection of estrogenic activity in municipal wastewater effluent using primary cell cultures from three-spined stickleback and chemical analysis.

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.

Atypical Aeromonas salmonicida Associated with Ulcerated Flatfish Species in the Baltic Sea and the North Sea

Abstract Skin ulceration in flatfish is a well-known clinical condition described in several reports since the beginning of the 20th century, but its etiology has not been fully described. In the present study European flounder Platichthys flesus, dab Limanda limanda, and plaice Pleuronectes platessa were examined for bacteria. The fish were from the southern Baltic Sea off the German and Danish coasts and from the North Sea off the Danish coast. Slow-growing, pinpoint colonies were isolated from skin ulcers of 73% of the fish examined. In one case, similar colonies were also isolated from the kidney. Based on growth characteristics and morphological, biochemical, and serological properties, the isolated bacteria were classified as “atypical” Aeromonas salmonicida. The strains isolated from ulcerated European flounder in this study seemed to be identical to the oxidase-negative strains previously isolated from European flounder off the Finnish coast. The strains isolated from dabs and plaice differed from...

Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum