Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene.

AIMS: Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. METHODS AND RESULTS: Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. CONCLUSIONS: The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. SIGNIFICANCE AND IMPACT OF THE STUDY: New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.

The effect of dietary phosphorus deficiency on the immune responses of European whitefish (Coregonus lavaretus L.).

Low phosphorous (P) feeds in aquaculture are recommended to reduce eutrophication of water systems. However, the feed should be adequate for normal growth and intact immune defence. Influence of low dietary P supply on non-specific and specific immune defence of European whitefish (Coregonus lavaretus) was studied in two trials. For Trial 1, a semi-purified, low-P diet was formulated and supplied with 0, 2.5, 5.0, 7.5, or 10.0 g P kg(-1)to obtain a P gradient of 4.4-14.9 g kg(-1)diet. Diets were fed to four replicate groups of fingerling whitefish for 42 days in a flow-through, freshwater system maintained at 15 degrees C. Fish fed with the P-unsupplemented diet had significantly lower plasma immunoglobulin M (IgM) levels than fish fed with the P-fortified diets (means 2.53 vs. 3.19 mg ml(-1); P=0.047). Plasma lysozyme activity did not differ between fish fed with the P-unsupplemented diet and fish fed with the P-fortified diets (means 21.8 vs. 25.5 U ml(-1); P=0.107). For Trial 2, fish were acclimated for 49 days by feeding the lowest or highest dietary P contents, and thereafter immunised with a single intraperitoneal injection of a novel antigen, bovine gamma globulin (BGG). A clear antibody response against BGG was elicited but the antibody levels did not differ between fish fed low-P or high-P contents. Similar to Trial 1, plasma IgM was lower in fish fed low-P diets, and low-P diet did not influence plasma lysozyme activity. In both trials the growth of fish, having received low-P diet, was significantly lowered. It is concluded that P deficiency has only minor effects on the immune parameters in whitefish, and the practical aquafeed with P contents sufficient for normal growth does not compromise immune functions of this species.

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). Part III: Lymphocytes.

The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a T-cell-specific mitogen, concanavalin A (ConA), or a B-cell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVB-exposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine gamma-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyte-related functions in fish.

Comparative effects of UVA and UVB irradiation on the immune system of fish.

Aquatic organisms can be harmed by the current levels of solar ultraviolet radiation. We have recently shown that exposure of fish to UVB irradiation alters the functioning of the fish immune system, but the effects of UVA radiation are unknown. The present study continues this work by characterizing UVA irradiation-induced immunological changes in fish. Roach, a cyprinid fish, were exposed to a single dose of either UVA (3.6 J/cm2) or UVB (0.5 J/cm2) irradiation. Both irradiations suppressed transiently mitogen-stimulated proliferation of blood lymphocytes. UVA, but not UVB, decreased hematocrit, plasma protein, and plasma immunoglobulin levels and increased the proportions of blood cells classified as unidentified leukocytes, possibly consisting of UVA-damaged lymphocytes. UVB, but not UVA, altered the functioning of head kidney and blood phagocytes, induced granulocytosis and lymphocytopenia in the blood and increased plasma cortisol concentration. These results imply that both UVA and UVB are potent modulators of the immune defence of fish.

Effects of primary- and secondary-treated bleached kraft mill effluents on the immune system and physiological parameters of roach.

The present study was designed to examine, whether, effluents from a modern pulp and paper mill using elemental chlorine-free/total chlorine-free (ECF/TCF) bleaching, exert effects on the immune system of fish and, in addition, to relate these findings to physiological parameters known to be affected by bleached kraft-mill effluents (BKME). Roach (Rutilus rutilus) were exposed in laboratory conditions to primary- or secondary-treated effluent from a pulp and paper mill. In order to study their capability to respond to foreign antigens they were immunised with bovine gamma-globulin (BGG) prior to exposure. The number of anti-BGG antibody-secreting cells (ASC) and the number of immunoglobulin-secreting cells (ISC) in the spleen and blood as well as the level of anti-BGG specific antibodies and concentration of plasma immunoglobulin (IgM) were studied. Phagocytosis and migration of granulocytes of the head kidney were also determined. In addition to the immunological parameters, the activity of hepatic biotransformation enzymes, the carbohydrate metabolism and osmoregulation were examined. Exposure of roach for 21 days to BKME affected several immunological parameters. Both effluents, primary- and secondary-treated, impaired the immunoreactivity of the fish. Sex-related differences in the immune responses were evident in many parameters e.g. in the number of blood ISC and splenic ASC. Sex also had effects on cortisol levels and in the induction of 7-ethoxyresorufin O-deethylase (EROD). These results demonstrate that both primary- and secondary-treated effluent from a pulp and paper mill using ECF/TCF bleaching have effects on fish immune functions. Further, these findings suggest that steroids may contribute to immunomodulation in fish.

Effects of ultraviolet light on immune parameters of the roach.

Ultraviolet B radiation penetrates into water and can affect fish health and the immune system, as is the case with mammals. Teleost fish, the roach, were exposed to UVB irradiation in aquariums and a panel of immune parameters was determined. In addition to altered blood picture and respiratory burst by blood leukocytes, changes were noted also in major lymphatic organs. Respiratory burst and natural cytotoxicity activity of head kidney granulocytes and mitogen-activated proliferation of splenic lymphocytes were suppressed. Although mostly transitory, some parameters remained suppressed for the following 2 weeks. Ultraviolet A radiation had only minor effects. The stress induced by UVB may be involved in the modulation of immune parameters.

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). II: Blood.

The effects of a single dose of ultraviolet B (UVB) radiation (0.4 J/cm2) on immunological functions by blood leukocytes and on hematological parameters was studied in roach (Rutilus rutilus), a teleostean fish. The respiratory burst of phorbol 12-myristate 13-acetate stimulated whole blood phagocytes increased significantly after UVB irradiation but spontaneous cytotoxicity of blood leukocytes toward 51chromium-labeled K562 target cells was not markedly altered. Differential cell counting revealed that UVB exposure significantly increased the proportion of granulocytes and significantly decreased the proportion of lymphocytes in the peripheral blood, whereas hematocrit and the total number of white and red blood cells were unchanged. Plasma cortisol concentration increased in UVB-exposed fish. Severe handling stress caused similar, although not as potent, effects on the measured parameters of fish blood as UVB irradiation. These observations suggest that in fish UVB brings about a stress response, which may account for the observed alterations in the immune parameters and leukocyte composition of blood. Exposure of fish to strong visible light induced no alterations in immunological or hematological parameters, making it unlikely that ultraviolet radiation mediates its effects through visual perception.

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). I. Phagocytes.

Roach (Rutilus rutilus) were irradiated with a single dose of ultraviolet B (UVB) radiation (0.4 J/cm2) in order to study the effects of UVB on the nonspecific immune defense mechanisms of fish. Neutrophils and macrophages were isolated from the head kidney of fish on days 1-14 postirradiation. Both random and directed migration of neutrophils, studied by migration under agarose assay, were suppressed on day 1 after UVB irradiation. The respiratory burst of phorbol 12-myristate 13-acetate-stimulated neutrophils and macrophages was also suppressed at days 1 and 2 after UVB irradiation. The suppression of migration and respiratory burst were restored or the responses were even enhanced later, but on the other hand spontaneous cytotoxicity of neutrophils toward 51chromium-labeled K562 target cells stayed suppressed throughout the 14 day follow-up. This study indicates that UVB radiation has the potential to suppress the functioning of phagocytes and to compromise the immune system of fish.

Immunoreactivity of roach, Rutilus rutilus, following laboratory exposure to bleached pulp and paper mill effluents.

In order to study immunomodulation, controlled laboratory experiments were carried out with roach (Rutilus rutilus) exposed to bleached kraft mill effluent (BKME) or spent bleach liquor (SBL) from two pulp and paper mills using elementary chlorine and chlorine dioxide for bleaching. The total number of immunoglobulin-secreting cells (ISC) and the number of specific antibody-secreting cells (ASC) were determined by ELISPOT assay. Exposure to BKME resulted in decreased numbers of ISC in the spleen. To study the capability of response against foreign antigens the fish were immunized with bovine gamma-globulin. Exposure to BKME or SBL reduced antigen-specific ASC response in fish. Moreover, the suppression of ASC response did not require exposure prior to immunization. Depuration of fish in clean water reveals that the immunosuppression caused by BKME is reversible. The ASC response in the BKME-pretreated fish returned to the same or even a higher level compared to untreated immunized fish and, in addition, the number of ISC increased greatly. The results of this study verify an earlier finding on poor antibody response due to exposure in a lake contaminated with BKME. Exposure to SBL alone suppresses antibody-mediated immunity, suggesting that compounds formed in bleaching are at least partly responsible for immunotoxic effects.

Subchronic effects of pulp and paper mill effluents on the immunoglobulin synthesis of roach, Rutilus rutilus.

Roach (Rutilus rutilus), from a unpolluted lake, were caged in a lake receiving treated waste waters from a pulp and paper mill 16 km upstream, and simultaneously a group of fish were held in cages in the unpolluted reference lake. The level of serum immunoglobulin of the fish kept in cages in these lakes was quantified. Serum immunoglobulin levels decreased when the fish were transferred to the contaminated lake and remained significantly reduced for the 8-week period fish were monitored. In another experiment fish were immunized with bovine gamma-globulin (BGG) after 5 weeks caging in the lakes. The kinetics of specific antibody synthesis were determined by assaying the number of antibody secreting cells in the spleen using enzyme-linked immunospot assay and by measuring the concentration of specific antibody in the serum. The peak response of the effluent exposed fish against BGG was lower than that for fish kept in the unpolluted reference lake, and the antibody titers, due to immunization, increased more slowly in the exposed fish. These results demonstrate that subchronic exposure of fish in a lake receiving treated waste waters from a bleached kraft mill affects the antibody-mediated immunity resulting in, besides lower levels of Ig, weak responsiveness against antigen.

Prediction of severe rheumatoid arthritis using Epstein-Barr virus.

One of the immunological abnormalities in patients with RA is increased synthesis of immunoglobulins (Ig) in cultures of Epstein-Barr virus (EBV-) stimulated lymphocytes. We set out to investigate whether this feature, seen early in the disease, associated with later severe RA. We studied prospectively 45 patients with recent onset RA and, 41 healthy individuals. From 0-6 months after admission, blood lymphocytes were cultured in the presence of EBV for 4 weeks and Ig in the supernatants were assayed. To assess the severity of disease, clinical, laboratory and radiological evaluations were performed every 6 months for 2 yr. The association of increased Ig production with the severity of RA was then analysed. During the follow-up period, 30 of the original 45 patients developed erosive disease. At onset, these 30 patients did not differ from the 15 with non-erosive disease when assessed by several parameters reflecting rheumatoid activity. However, EBV-induced production of Ig was significantly higher in the erosive compared with the non-erosive group of patients (P < 0.001). Using Ig synthesis, it was possible to identify a subgroup of 9-14 patients, depending on the isotype studied, who would later develop severe erosive disease (PVpos = 90-100%). These results show that high EBV-induced production of Ig early in RA associates with later severe disease, particularly with joint erosions. This feature identifies, with over 90% likelihood, the third of patients who will later develop most severe disease.

Association of in vitro immune functions with the severity of the disease in rheumatoid arthritis.

The production of immunoglobulins in vitro by lymphocytes from rheumatoid patients has been earlier shown to be defective. This report describes a 2-year follow up study which shows that this defect is associated with the severity of RA. Pokeweed mitogen and Staphylococcus aureus Cowan I were used to stimulate in vitro immunoglobulin production by lymphocytes from patients with recent onset RA, and the relationship of responses to clinical characteristics were studied. Impaired polyclonal IgM synthesis, already detectable at the onset of disease, associated with joint destructions observed after a 2-year follow up period. Further, phytohaemagglutinin-induced interleukin-2 (IL-2) release by the cells of patients with erosive disease was found to be reduced compared to cells from patients without eroded joints. The results indicate that altered immune functions--manifested as decreased production of IgM and IL-2--in RA are involved in the progression of the disease and affect the outcome of patients and, thus, represent an unfavourable prognostic feature.