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 immunoenzymatic a nd immunofluorescence 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, b oth specific for the 57-58-kD outer membrane protein (p57) of the bact erium. Both MAbs revealed positive staining in ethanol-fixed tissue sp ecimens, but only the epitope identified by MAb 4D3 was formalin resis tant. Pretreatment with trypsin did not reestablish the antigenicity f or 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 syste m was obtained after serial dilution of the Mab (2G5) or the chromagen , amino ethyl carbazole, in the first sequence. Paired immunofluoresce nce staining with well-balanced color mixing was easily obtained on et hanol-fixed tissue specimens using sequential incubations. Single expo sures gave blue (aminomethyl coumarin acetic acid) and green (fluoresc ein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, re spectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount o f a protein preparation of p57 into an inert matrix, creating an artif icial test substrate. Antigen detection sensitivity based on an immuno enzymatic alkaline phosphatase method and Fast Red as chromagen and ev aluated by an image-analysis system creating a relative grey-level sco re showed an 8-fold higher detection sensitivity on ethanol-fixed as c ompared with formalin-fixed specimens. Monoclonal antibodies specific for the outer membrane protein of Renibacterium salmoninarum are well suited for use in immunohistochemical techniques for in situ identific ation of the bacterium in paraffin-embedded tissue specimens. Serial e valuation of paired stained sections with a confirmatory diagnosis of BKD being dependent on both MAbs giving a positive reaction would incr ease the specificity of the test. Simultaneous identification of the t wo epitopes of p57 on the same section was dependent on ethanol-fixed tissue specimens.