Abstract
To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aedo, E. & Bustos, P. 1991 Obtención de ovas de salmones libres de BKD. Chile Pesquero 62, 51–55.
Austin, B., Bucke, D., Feist, S. & Rayment, J.N. 1985 A false positive reaction in the fluorescent antibody test for Renibacterium salmoninarum with a ‘coryneform’ organism. Bulletin of the European Association of Fish Pathologists 5, 8–9.
Austin, B., Embley, T.M. & Goodfellow, M. 1983 Selective isolation of Renibacterium salmoninarum. FEMS Microbiology Letters 17, 111–114.
Brown, L.L., Ricks, R., Evelyn, T.P.T. & Albright, L.J. 1990 Experimental intra-ovum infection of coho salmon (Orcorhynchus kisutch) eggs with Renibacterium salmoninarum using a microinjection technique. Diseases of Aquatic Organisms 8, 7–11.
Bruno, D.W. 1987 Serum agglutinating titers against Renibacterium salmoninarum, the causative agent of bacterial kidney disease, in rainbow trout Salmo gairdneri Richardson and Atlantic salmon, Salmo salar L. Journal of Fish Biology 30, 327–334.
Bullock, G.L., Griffin, B.R. & Stuckey, H.M. 1980 Detection of Corynebacterium salmoninus by direct fluorescent antibody test. Canadian Journal of Fisheries and Aquatic Science 37, 719–721.
Bullock, G.L. & Stuckey, H.M. 1975 Fluorescent antibody identification and detection of the Corynebacterium causing kidney disease of salmonids. Journal of the Fisheries Research Board of Canada 32, 2224–2227.
Chien, M.S., Gilbert, T.L., Huang, C., Landolt, M.L., O'Hara, P.J. & Winton, J.R. 1992 Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum. FEMS Microbiology Letters 96, 259–266.
Cipriano, R.C., Starliper, C.E. & Schachte, J.H. 1985 Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes. Journal of Wildlife Diseases 21, 1441–1448.
Daly, J.G. & Stevenson, R.M.W. 1985 Charcoal agar, a new growth medium for the fish disease Renibacterium salmoninarum. Applied and Environmental Microbiology 50, 868–871.
Dixon, P.F. 1987 Comparison of serological techniques for the identification of Renibacterium salmoninarum. Journal of Applied Icthyology 3, 131–138.
Etchegaray, J.P., Martínez, M.A., Krauskopf, M. & León, G. 1991 Molecular cloning of Renibacterium salmoninarum fragments. FEMS Microbiology Letters 79, 61–64.
Evelyn, T.P.T. 1977. An improved growth medium for the kidney disease bacterium and some notes on using the medium. Bulletin de l'Office International des Epizooties 87, 511–513.
Evelyn, T.P.T., Ketcheson, J.E. & Prosperi-Porta, L. 1981 The clinical significance of immunofluorescence-based diagnoses of the bacterial kidney disease carrier. Fish Pathology 15, 293–300.
Evelyn, T.P.T., Ketcheson, J.E. & Prosperi-Porta, L. 1984 Further evidence for the presence of Renibacterium salmoninarum in salmonid eggs and for the failure of povidone-iodine to reduce the intra-ovum infection rate in water-hardened eggs. Journal of Fish Diseases 7 173–182.
Evenden, A.J., Gilpin, M.L. & Munn, C.B. 1990 The cloning and expression of a gene encoding haemolytic activity from the fish pathogen Renibacterium salmoninarum. FEMS Microbiology Letters 71, 31–34.
Fryer, J.L. & Sanders, J.E. 1981 Bacterial kidney disease of salmonid fish. Annual Review of Microbiology 35, 273–298.
Gutenberger, S.K., Giovannoni, S.J., Field, K.G., Fryer, J.L. & Rohovec, J.S. 1991 A phylogenetic comparison of the 16S rRNA sequence of the fish pathogen, Renibacterium salmoninarum, to Gram-positive bacteria. FEMS Microbiology Letters 77, 151–156.
Humphrey, J.D., Lancaster, C.E., Gudkovs, N. & Coplands, J.W. 1987 The disease status of Australian salmonids: bacteria and bacterial diseases. Journal of Fish Diseases 10, 403–410.
Kohne, D.E., Levison, S.A. & Byers, M.J. 1977 Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique. Biochemistry 16, 5329–5341.
Kunkel, L.M., Monaco, A.P., Middlesworth, W., Ochs, H.D. & Latt, S.A. 1985 Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proceedings of the National Academy of Sciences of the United States of America 82, 4778–4782.
Lee, E.G.H. & Gordon, M.B. 1987 Immunofluorescence screening of Renibacterium salmoninarum in the tissues and eggs of farmed chinook salmon spawners. Aquaculture 65, 7–14.
McCabe, J.T. & Pfaff, D.W. 1989 In situ hybridization: a methodological guide. Methods in Neurosciences 1, 98–126.
Pascho, R.J. & Mulcahy, D. 1987 Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Canadian Journal of Fisheries and Aquatic Sciences 44, 183–191.
Sakai, M., Amaaki, N., Atsuta, S. & Kobayashi, M. 1987 Comparative sensitivities of several dot blot methods used to detect bacterial kidney disease. Journal of Fish Diseases 10, 229–231.
Sanders, J.E. & Barros, J. 1986 Evidence by the fluorescent antibody test for the occurrence of Renibacterium salmoninarum using salmonid fish in Chile. Journal of Wildlife Diseases 22, 255–257.
Sanders, J.E. & Fryer, J.L. 1988 Bacteria of Fish. In Methods in Aquatic Bacteriology ed Austin, B. pp. 115–142. Chichester: John Wiley & Sons.
Sanger, F., Nicklen, S. & Coulson, A.R. 1977 DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America 74, 5463–5467.
Singer, R.H., Lawrence, J.B. & Raschtchian, R.N. 1989 Toward a rapid and sensitive in situ hybridization methodology using isotopic and nonisotopic probes. Methods in Neurosciences 1, 71–96.
Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503–517.
Trust, T.T. 1986 Pathogenesis of infectious diseases of fish. Annual Review of Microbiology 40, 479–502.
Vega, I., León, G., Muñoz, R.I., Zapata, C. & Krauskopf, M. 1990 Utilización de una sonda de DNA especifica en la confirmación de anemia falciforme en una mujer caucasiana. Revista Médica de Chile 118, 306–312.
Walker, J. & Dougan, G. 1989 DNA probes: a new role in diagnostic microbiology. Journal of Applied Bacteriology 667, 229–238.
Additional information
The authors are with the instituto de Bioquímica, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile
Rights and permissions
About this article
Cite this article
León, G., Martinez, M.A., Etchegaray, J.P. et al. Specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum . World Journal of Microbiology & Biotechnology 10, 149–153 (1994). https://doi.org/10.1007/BF00360876
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00360876