Modulatory effects of cadmium and copper on the susceptibility and immune response of common carp, Cyprinus carpio (L) to selected pathogens

Abstract

Pollution of the aquatic environment has received considerable attention in recent years. The fear that pollutants might predispose fish to disease is a cause of great concern. Recent evidence has shown that, in the case of some pollutants, the fear was well-founded. The lack of detailed studies in this field prompted the present study. A comprehensive approach was made towards understanding the effects of two common aquatic pollutants, the metals cadmium and copper, at sublethal levels, on the disease resistance and immune response mechanisms of common carp, Cyprinus carpio L. Two pathogens were used as models, which differ from each other with respect to the nature of the disease processes and their cellular requirements for optimal immune response. Ichthyophthirius multifiliis (Fouquet), the causative agent of white spot disease and Aeromonas hydrophila which causes bacterial haemorrhagic septicaemia were employed to examine the immunomodulatory effects of the metals. A third model, Sheep Red Blood Cell, with known cellular requirements and immunokinetics, was used as a reference antigen. The in vitro and in vivo effects of the metals on lymphocyte proliferation induced by putative T and B-cell mitogens were also evaluated. The ability of the metals to induce a stress response in carp was assessed by monitoring the kinetics of plasma cortisol. Subtle structural changes produced by the metals in the primary barriers and haematopoietic organs were also examined histologically. Exposure of naïve carp for 10 days to sublethal levels (50****) of cadmium and copper significantly increased their susceptibility to I. multifiliis as measured by mean parasite intensity. Previously immunized carp with established immunity and high “tomite agglutination titre” could not mount a protective immune response following 10 days exposure to 25 and 50 **** cadmium and copper. Concurrent exposure of carp to either cadmium or copper with a series of controlled low level immunization exposures to the parasite, did not alter the kinetics nor the magnitude of the “anti-ich” humoral antibody titre. Control carp acquired complete resistance earlier than metal exposed ones. Exposure of carp to cadmium (50 *** *) or copper (30 ****) for 40 days with the primary immunization given 10 days after the commencement of metal exposure did not reduce the primary antibody response, but significantly reduced the magnitude of the secondary response. Similar experimental conditions also revealed die significant suppressive effects of these metals on the number of both Rosette Forming and Plaque Forming Cells. Long term exposure (30 days) to cadmium before the primary immunization did not impair the kinetics and magnitude of the primary and secondary humoral response, but, in contrast, copper significantly suppressed both the primary and secondary response. Exposure of carp to the metals 18 days after the primary immunization with SRBC significantly reduced the magnitude of the secondary humoral response. Primary immunization given concurrently with the commencement of metal exposure, significantly reduced the magnitude of both the primary and secondary response. Carp exposed to cadmium and copper for 10 days were more susceptible to A. hydrophila than unexposed controls. Previously immunized carp could not mount an effective protective immunity following 10 days exposure. In contrast to I. multifiliis, the decrease in the protective immunity was associated with a significant drop in the humoral bacterial agglutination titre. Exposure to the metals for 10 days before immunization with A. hydrophila heat killed bacterin reduced the magnitude of the humoral reprise significantly. Immunization carried out simultaneously with the commencement of metal exposure suppressed the magnitude of both the primary and secondary humoral antibody response. Lymphocytes collected from carp exposed to cadmium (50 Ugl ') or copper (30 ****) showed a reduced blastogenic response to the mitogens concanavalin (Con A) and lipopolysaccharide (LPS). A significant suppression was observed in lymphocytes collected after 6 to 9 days of exposure to the respective metals. Both the metals in vitro showed similar suppressive effects. Concentrations of 10**M (copper) or 10**M (cadimum) and greater, suppressed the mitogenic response to both the mitogens significantly. Exposure to both the metals induced a stress response, characterized by elevation of plasma cortisol. The elevation in cadmium expired carp was transitory in nature, the values returning to near basal values and persisting at that level till the end of the experimental duration. In the copper exposed carp, the cortisol decreased gradually from the maximum seen following 24 hours until day 9 before showing a secondary elevation which persisted. Both the metals produced leucopenia. Cadmium exposure did not have any effect on the haematocrit, whilst copper caused an elevation. The pathological effects on the structure of the primary barriers were considerable at the concentrations tested. The pathological changes in the haematopoietic organs were not so severe, but included multifocal necrosis of the haematopoietic tissue and indications of fragmentation of malanomacrophage centres in the kidney and spleen. Both cadmium (50 ****) and copper (30 ****) did not completely abrogate the immune response of carp. Studies evaluating the immunotoxicity of aquatic pollutants should give more emphasis to the magnitude of suppression rather than looking for a complete abrogation of immune response. This study has clearly shown that a reduction in the magnitude of the immune response is sufficient to predispose fish to disease. These findings are significant when assessing the impact of these pollutants and when determining the maximum limits tolerable in the natural environment.