Serological and genetic characterization of Flavobacterium psychrophilum isolated from farmed salmonids in Turkey.

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.

In vivo adherence of Flavobacterium psychrophilum to mucosal external surfaces of rainbow trout (Oncorhynchus mykiss) fry.

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.

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.

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.

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.

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.

Immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), with a low molecular mass fraction isolated from Flavobacterium psychrophilum.

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.

Estrogen- and androgen-sensitive bioassays based on primary cell and tissue slice cultures from three-spined stickleback (Gasterosteus aculeatus).

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback (Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.

Cost-effectiveness of intensive adjuvant chemotherapy for high-risk breast cancer: is tailored and dose-escalated chemotherapy with growth factor support (GFS) more costly and less effective than marrow-supported high-dose chemotherapy - results from a randomized study.

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, p <0.001), 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, p <0.001). 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.

Long-term follow-up of the SBG 9401 study comparing tailored FEC-based therapy versus marrow-supported high-dose therapy.

BACKGROUND: The purpose was to investigate adjuvant marrow-supportive high-dose chemotherapy compared with an equitoxicity-tailored comparator arm. PATIENTS AND METHODS: Five hundred and twenty-five women below the age of 60 years with operated high-risk primary breast cancer were randomised to nine cycles of granulocyte colony-stimulating factor supported and individually tailored FEC (5-fluorouracil, epirubicin, cyclophosphamide), (n = 251) or standard FEC followed by marrow-supported high-dose therapy with CTCb (cyclophosphamide, thiotepa, carboplatin) therapy (n = 274), followed by locoregional radiotherapy and tamoxifen for 5 years. RESULTS: There were 104 breast cancer relapses in the tailored FEC group versus 139 in the CTCb group (double triangular method by Whitehead, P = 0.046), with a median follow-up of all included patients of 60.8 months. The event-free survival demonstrated 121 and 150 events in the tailored FEC- and CTCb group, respectively [P = 0.074, hazard ratio (HR) 0.804, 95% confidence interval (CI) 0.633-1.022]. Ten patients in the tailored FEC regimen developed acute myeloid leukaemia (AML)/myelodysplasia (MDS). One hundred deaths occurred in the tailored FEC group and 121 in the CTCb group (P = 0.287, HR 0.866, 95% CI 0.665-1.129). CONCLUSION: The update of this study shows an improved outcome linked to the tailored FEC treatment in relation to breast cancer relapse, but also an increased incidence of AML/MDS.

Autologous stem cell transplantation in elderly (>60 years) patients with non-Hodgkin's lymphoma: a nation-wide analysis.

Limited experience is available on the feasibility and efficacy of autologous stem cell transplantation (ASCT) in elderly patients with non-Hodgkin's lymphoma (NHL). In 1994-2004 altogether 88 NHL patients > 60 years old received ASCT in six Finnish transplant centres. There were 57 male and 31 female patients with a median age of 63 years (range 60-70 years); 17 patients were>65 years. The histology included diffuse large B cell (n = 29), mantle cell (n = 27), follicular (n = 15), peripheral T cell (n = 12) and other (n = 5). Disease status at ASCT was I complete remission/partial remission (CR/PR) in 53 patients, II CR/PR in 30 patients and other in five patients. The conditioning regimens included BEAC (n = 49), BEAM (n = 34), TBI-CY (n = 4) and other (n = 1). Eighty-four patients received PB grafts. The medians to reach neutrophils > 0.5 and platelets > 20 were 10 and 14 days, respectively. The early treatment-related mortality (TRM) (<100 days) was 11%. With a median follow-up of 21 months for all patients, 45 patients (51%) are alive. A relapse or progression after ASCT has been observed in 32 patients (36%). ASCT is feasible in selected elderly patients with NHL, but the early TRM seems to be higher than in younger patients.

Early treatment-related mortality in adult autologous stem cell transplant recipients: a nation-wide survey of 1482 transplanted patients.

OBJECTIVES: To evaluate early (<100 d) treatment-related mortality (TRM) in autologous stem cell transplant (ASCT) recipients. PATIENTS: Altogether 1482 adult patients received ASCT in six Finnish centres 1990-2003. The most common diagnoses were non-Hodgkin's lymphoma (NHL) (n = 542), multiple myeloma (MM) (n = 528), breast cancer (BC) (n = 132), Hodgkin's lymphoma (n = 86) and chronic lymphocytic leukaemia (CLL) (n = 63). RESULTS: Forty-two patients (2.8%) died from treatment-related reasons <100 d from ASCT. The median time to death was 38 d from ASCT (0-99). The risk of TRM varied according to the diagnoses. The highest risk was observed in patients with AL amyloidosis (24%) followed by NHL (4.4%) and MM (1.9%). No early TRM was observed in patients transplanted for BC or CLL. Infections were the cause of death in 16 patients (fungal 7, bacterial 6, viral 3). Organ toxicity was responsible for early death in 26 patients (heart 9, lungs 7, other 10). CONCLUSIONS: This nation-wide survey indicated a low early TRM in ASCT recipients in general, but higher risks in patients with AL amyloidosis or NHL. In addition to patient selection, also optimization of transplant procedure may be needed in these patient groups to reduce early TRM.

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum).

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.

Outcome of progressive disease after autologous stem cell transplantation in patients with non-Hodgkin's lymphoma: a nation-wide survey.

OBJECTIVES: To analyse outcome and prognostic factors in non-Hodgkin's lymphoma (NHL) patients who progress after autologous stem cell transplantation (ASCT). PATIENTS: Altogether 115 consecutive NHL patients transplanted in 1991-2000 were studied. Histology included diffuse large B cell (n = 52), follicular (n = 26), mantle cell (n = 15), T cell (n = 16) and other subtypes (n = 6). The median time from ASCT to the progression was 7 months. Ninety-six patients (83%) received salvage treatment. RESULTS: Twenty-four patients (25%) achieved complete remission and 30 (31%) partial remission. The median overall survival was 8 months (range 0-98+) and the projected 4-year survival 21%. In multivariate analysis factors predicting treatment response after the progression included the use of rituximab (P = 0.036), histology other than diffuse large B cell (P = 0.001) and International Prognostic Index < or =2 at progression (P < 0.001). Normal lactate dehydrogenase (LDH) at progression (P = 0.002), response to salvage treatment (P < 0.001) and time from ASCT to progression > or =7 months (P = 0.022) were predictors for overall survival. CONCLUSIONS: Although the prognosis of patients who progress after ASCT is generally poor, many patients will respond to current therapies, and some may experience prolonged survival. Normal LDH at time of disease progression and longer time to progression after ASCT were the most powerful predictors for a promising outcome.

Invasive fungal infections in autologous stem cell transplant recipients: a nation-wide study of 1188 transplanted patients.

UNLABELLED: Based on small single-centre series, the risk of invasive fungal infections (IFI) has been considered small in autologous stem cell transplant (ASCT) recipients. PURPOSE: To analyse epidemiological and clinical features of (IFI) among ASCT recipients in Finland 1990-2001. PATIENTS: During the study period, 1188 adult patients received high-dose therapy supported by ASCT in six centres. Altogether, 1112 patients (94%) received blood progenitor cells. The graft was CD34+ selected in 261 patients (22%). The major diagnostic groups were non-Hodgkin's lymphoma (n = 417), multiple myeloma (n = 395), breast cancer (n = 132) and Hodgkin's lymphoma (n = 53). RESULTS: Eighteen patients (1.5%) with IFI were identified. The incidence of proven or probable invasive aspergillosis was 0.8%, followed by candidaemia with an incidence of 0.3%. The median time to the diagnosis of IFI was 35 d (6-162) from the progenitor cell infusion. In fourteen patients (78%) IFI was diagnosed during lifetime and they were treated with antifungal therapy for a median of 50 d. Nine patients (64%) were cured. CONCLUSIONS: IFI appears to be a rare event after ASCT and Aspergillus infections seem to be predominant. These epidemiological features have an impact in planning prophylactic and empirical antifungal strategies in ASCT recipients.

Autologous stem cell transplantation in adult patients with peripheral T-cell lymphoma: a nation-wide survey.

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.

Scandinavian Sarcoma Group Osteosarcoma Study SSG VIII: prognostic factors for outcome and the role of replacement salvage chemotherapy for poor histological responders.

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 < 190 ml, 24-h serum methotrexate > 4.5 microM 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.