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

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.

References

Arakawa

Sanders

Fryer

: 1987, Production of monoclonal antibodies against Renibacterium salmoninarum. J Fish Dis 10:249–253.

Austin

: 1987, Aerobic gram-positive rods. In: Bacterial fish pathogens: disease in farmed and wild fish, ed. Laird

, pp. 70–87. Ellis Horwood, New York, NY.

Austin

Embley

Goodfellow

: 1983, Selective isolation of Renibacterium salmoninarum. FEMS Microbiol Lett 17:111–114.

Avrameas

Ternynck

: 1969, The crosslinking of proteins with gluataraldehyde and its use for preparation of immunoadsorbents. Immunochemistry 6:53–66.

Brandtzaeg

: 1972, Evaluation of immunofluorescence with artificial sections of selected antigenicity. Immunology 22:177–183.

Brandtzaeg

: 1982, Tissue preparation methods for immunohistochemistry. In: Techniques in immunohistochemistry, ed. Bullock

Petrusz

, pp. 2–75. Academic Press, London, England.

Brandtzaeg

Rognum

: 1984, Evaluation of nine different fixatives. 2. Preservation of IgG, IgA and secretory component in an artificial immunohistochemical test substrate. Histochemistry 81:213–219.

Bullock

Stuckey

: 1975, Fluorescent antibody identification and detection of the Corynebacterium causing kidney disease of salmonids. J Fish Res Board Can 32:2224–2227.

Daly

: 1989, Growth and cell surface studies of Renibacteriurn salmoninarum. PhD Thesis, University of Guelph, Guelph, ON, Canada.

10.

Daly

Stevenson

RMW

: 1985, Charcoal agar, a new growth medium for the fish disease bacterium Renibacterium salmoninarum. Appl Environ Microbiol 50:868–871.

11.

Evelyn

TPT

Ketcheson

Properi-Porta

: 1984, Further evidence for the presence of Renibacterium salmoninarum in salmonid eggs and for the failure of providone-iodine to reduce the intra-ovum infection rate in water-hardened eggs. J Fish Dis 71:173–182.

12.

Evensen

: 1993, An immunohistochemical study on the cytogenetic origin of pulmonary multinucleate giant cells in porcine dermatosis vegetans. Vet Pathol 30:162–170.

13.

Ferguson

: 1989, Bacterial kidney disease. In: Systematic pathology of fish, pp. 78–79. Iowa State University Press, Ames, IA.

14.

Fox

Johnson

Whiting

Roller

: 1985, Formaldehyde fixation. J Histochem Cytochem 33:845–853.

15.

Haines

Chelack

: 1991, Technical considerations for developing enzyme immunohistochemical staining procedures on formalin-fixed paraffin-embedded tissues for diagnostic pathology. J Vet Diagn Invest 3:101–112.

16.

Hed

Enestrom

: 1981, Detection of immune deposits in glomeruli: the masking effect on antigenicity of formalin in the presence of proteins. J Immunol Methods 41:57–62.

17.

Hoffmann

Bell

Pfeil-Putzien

Ogawa

: 1989, Detection of Renibacterium salmoninarum in tissue sections by different methods-a comparative study with special regard to the indirect immunohistochemical peroxidase technique. Fish Pathol 24:101–104.

18.

Høyer-Hansen

: 1988, Characterization of monoclonal antibodies. In: Handbook of immunoblotting of proteins, vol. II. Experimental and clinical applications, ed. Bjerrum

Heegaard

NHH

, pp. 1–12. CRC Press, Boca Raton, FL.

19.

Laemmli

: 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

20.

Martin

: 1977, The evaluation of tests. Can J Comp Med 41:19–25.

21.

Martin

Meek

Willeberg

: 1987, Veterinary epidemiology. Principles and methods, pp. 121–148. Iowa State University Press, Ames, IA.

22.

Mason

Sammons

: 1978, Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol 31:454–460.

23.

Mepham

Frater

Mitchell

: 1979, The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique. Histochem J 11:345–357.

24.

Miettinen

: 1989, Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity. Pathol Res Pract 184:431–436.

25.

Mitchum

Sherman

: 1981, Transmission of bacterial kidney disease from wild to stocked hatchery trout. Can J Fish Aquat Sci 38:547–551.

26.

Petrusz

: 1983, Essential requirements for the validity of immunocytochemical staining procedures. J Histochem Cytochem 31:177–179.

27.

Petrusz

Ordronneau

Finley

JCW

: 1980, Criteria of reliability for light microscopic immunohistochemical staining. Histochem J 12:333–348.

28.

Rockey

Gilkey

Wiens

Kaattari

: 1991, Monoclonal antibody-based analysis of the Renibacterium salmoninarum p57 protein in spawning chinook and coho salmon. J Aquat Anim Health 3:23–30.

29.

Rognum

Brandtzaeg

Ørjasaeter

Fausa

: 1980, Immunohistochemistry of epithelial cell markers in normal and pathological colon mucosa. Comparison of results based on routine formalin- and cold ethanol-fixation methods. Histochemistry 67:17–21.

30.

Takamyia

Bodemer

Vogt

: 1978, Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease. J Histochem Cytochem 26:914–920.

31.

Valnes

: 1989, Two-color immunohistochemistry for studies of immunological factors in human gastric mucosa, pp. 11–47. PhD Thesis, University of Oslo, Oslo, Norway.

32.

Valnes

Brandtzaeg

: 1984, Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels. Histochem J 16:477–487.

33.

Valnes

Brandtzaeg

Rognum

: 1984, Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections. Histochemistry 81:313–319.

34.

Wiens

Kaattari

: 1989, Monoclonal antibody analysis of common surface protein(s) of Renibacterium salmoninarum. Fish Pathol 24:1–7.

35.

Wiens

Kaattari

: 1991, Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum. Infect Immun 59:631–637.