Immunohistochemical identification of Renibacterium salmoninarum by monoclonal antibodies in paraffin-embedded tissues of Atlantic salmon (Salmo salar L.), using paired immunoenzyme and paired immunofluorescence techniques

Renibacterium salmoninarum was identified in situ by immunoenzymat ic and immunofluores- cence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin -peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate. Antigen detection sensitivity based on an immunoenzymatic alkaline phosphatase method and Fast Red as chromagen and evaluated by an image-analysis system creating a relative grey-level score showed an 8-fold higher detection sensitivity on ethanol-fixed as compared with formalin-fixed specimens. Monoclonal antibodies specific for the outer membrane protein of Renibacterium salmoninarum are well suited for use in immunohistoch emical techniques for in situ identification of the bacterium in paraffin-embe dded tissue specimens. Serial evaluation of paired stained sections with a confirmatory diagnosis of BKD being dependent on both MAbs giving a positive reaction would increase the specificity of the test. Simultaneous identification of the two epitopes of p57 on the same section was dependent on ethanol-fixed tissue specimens.