M. proteins targeted by immobilizing antibodies) in Freund’s complete adjuvant develop active protective immunity and produce antibodies against i-antigens (10, 16) in both the blood and the cutaneous mucus (4-6, 15, 45-47). Additionally, passive transfer to fish skin of immobilizing immunoglobulin G (IgG)-class murine monoclonal antibodies administered by intraperitoneal injection supports the concept that antibodies are a key component of the epithelial immune barrier (24). On the basis of this finding and on the basis of the observation that parasites rapidly leave the skin Y-33075 of as a model to investigate the cutaneous immune response of fish to pathogens that invade the fish through epithelial tissues (17). In this study, we used an enzyme-linked immunosorbent assay (ELISA) to compare over time the relative amounts of infection or the injection of purified antigen and that in both cases their occurrence did not exactly coincide with serum antibody production. Our results suggest that parasite-specific antibodies in the cutaneous mucus of channel catfish do not arise by passive transfer or exudation from the blood. MATERIALS AND METHODS Parasite propagation. The G5 isolate used in this study has been characterized previously, and its propagation by passage on channel catfish has been described (14). Purification of protein antigens. i-antigen was purified from isolate G5 serotype D theront membrane proteins by previously published methods (23). Aliquots were flash frozen in liquid nitrogen and stored at ?80C. Aliquots were thawed to room temperature (RT) and diluted in 25 mM sodium acetate Y-33075 (pH 7.5) immediately before use in the ELISA procedure. Detergent-extracted membrane protein was further Rabbit polyclonal to ADNP enriched for i-antigen by using a column on which a monoclonal antibody specific for G5 i-antigen (G-361) was immobilized as described previously (23). The immunoaffinity-purified i-antigen was used to inject fish by the intraperitoneal route. Production of anti-catfish Ig antibody. Ig was purified from pooled channel catfish (infection and formalin treatments to isolate specific groups of fish. Fish immunized by infection were kept in isolated aquaria with individual filter units until the infection was eliminated, at which time the fish were returned to their respective tanks. The water temperature ranged from 16 to 20C during a 2-month acclimatization period. The water temperatures ranged from 20 to 24C during the 14-week time course of the experiment. Immunization of fish with protein. Fish were anesthetized with tricaine methane-sulfonate (100 to 200 mg/liter; MS-222; Argent Chemicals, Redmond, Wash.) dissolved in water that had been buffered with equal amounts of sodium bicarbonate (Fisher). Each fish received 5.0 g of affinity-purified i-antigen diluted in 25 l of PBS and mixed 1:1 with Freund’s incomplete adjuvant. A 50-l volume was injected into the peritoneal cavity of each fish at the ventral surface midline by using a 1-ml tuberculin syringe (Monoject; Sherwood Medical Company, St. Louis, Mo.) fitted with a 23-gauge by 1-in. needle (Becton Y-33075 Dickinson & Co., Franklin Lakes, N.J.). Exposure of fish to parasites. Twenty catfish were exposed to theronts (isolate G5, serotype D) maintained by passage on channel catfish (14). Unanesthetized fish Y-33075 were placed 10 at a time in 2-liter plastic beakers filled with charcoal-filtered water (200 ml/fish) containing a known number of theronts at room temperature for 1 h. The fish were initially exposed to the theronts at a ratio of 5, 000 theronts/fish and were again exposed 2 weeks later at a ratio of 11,000 theronts/fish. Numerous parasites were observed on all fish 4 days after the second exposure. Formalin (formaldehyde, 37% solution; J. T. Baker, Phillipsburg, N.J.) was added to each tank at 0.26 ml/liter on the 5th day and every other day for 3 additional days. Complete water changes were performed twice a day starting on the evening of the 5th day and continuing for the duration of treatment. One fish died during treatment. The remaining fish appeared to be free of parasites 3 weeks after the infection was first observed. Cutaneous mucus and serum collection. Cutaneous mucus and blood samples collected from the fish 2 weeks prior to initiation of the experiment served as Y-33075 negative control (i.e., pretreatment) samples. After treatment, cutaneous mucus and serum samples were collected from the same fish at eight time points spanning a 14-week.