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

Abstract

Abstract. Renibacterium salmoninarum was identified in situ by immunoenzymatic and 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, 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 immunohistochemical techniques for in situ identification of the bacterium in paraffin-embedded 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.

Clinical Outbreaks of Bacterial Kidney Disease (BKD) in Hatchery-Raised Brook Trout (Salvelinus fontinalis) (Mitchill, 1814): Lessons Learned

“…In case of the Iron River BKT mortalities, IHC stained slides indicated the presence of an intense distribution of bacterial cells and their antigens within the kidney tissues, as well as within the granulomas, which confirms the assumption that the severe progression of the disease is associated with high bacterial metabolic activities [26][27][28]. Similar findings were reached by [14,15]. Some discrepancies were observed between histopathological findings (positive with different scores) and IHC results (negative results with some of positive histopathological changes).…”

“…In this context, Evensen et al [15] reported on their success in detecting R. salmoninarum in situ using paraffin embedded tissue specimens from infected Atlantic (Salmo salar). However, it has been reported that extended preservation of tissue samples in formalin has deleterious effect on the antigen detection and retrieval in IHC [14].…”

Comparison of immunohistochemistry and virus cultivation for detection of viral haemorrhagic septicaemia virus in experimentally infected rainbow trout Oncorhynchus mykiss

“…It has been shown that formalin fixation reduces the sensitivity of immunohistochemical techniques compared to precipitating fixatives, when using immune sera or monoclonal antibodies against protein epitopes , Valnes et al 1984. The differences in detection sensitivity, ex-pressed as the smallest amount of antigen revealed (Petrusz et al 1980), may range from 3-to 8-fold between ethanol and formalin fixation when evaluated in artificial antigen media , Valnes et al 1984, Evensen et al 1994; the adverse effects induced by fixation, especially by the aldehyde-containing fixatives, include the masking of tissue antigens with loss of antigenicity. The most recent previous report on in situ den~onstration of VHS virus describes a higher sensitivity of immunofluorescence methods compared to virus isolation when physical fixation and frozen sections are used (Ortega et al 1992).…”

The Use of Kidney Biopsy of Broodstock Steelhead Trout (Oncorhyncus Mykiss) to Determine the Status of Bacterial Kidney Disease Infection

“…The biopsy instrument b was placed into the surgical incision and blindly directed caudodorsally through the pericardial sac and into the cranialmost aspect of the kidney tissue. The biopsy forcep was then closed, removing a small sample of kidney tissue (approximately 1.0-1.5 mm 3 ). An impression smear was made of the fluid from the lateral surface of the biopsy forceps and examined microscopically for the presence of melanin granules to confirm the presence of renal tissue within the biopsy forceps (Fig.…”

“…Direct immunohistochemical procedures were performed as previously described, 3,5,10 Paraffin-embedded tissue sections were deparaffinized in an oven followed by 3 changes of toluene for 5 minutes each, 2 changes of 100% ethanol for 3 minutes each, and 2 changes of 95% ethanol for 3 minutes each. Slides were then rinsed in running water and incubated overnight in 5% hydrogen peroxide to remove melanin pigments.…”