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Applied and Environmental Microbiology, February 2001, p. 750-759, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.750-759.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Antigenic Characterization of the Fish Pathogen Flavobacterium psychrophilum

Elizabeth M. Crump,1 Malcolm B. Perry,2 Sharon C. Clouthier,1,3 and William W. Kay1,3,*

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6,1 National Research Council, Institute of Biological Sciences, Ottawa K1A 0R6,2 and Microtek International Ltd., Saanichton, British Columbia V8M 1Z8,3 Canada

Received 21 July 2000/Accepted 7 December 2000

Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide. The predominant ~16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant ~20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilum strains.

* Corresponding author. Mailing address: Department of Biochemistry and Microbiology, Petch Building, University of Victoria, P.O. Box 3055, Victoria, British Columbia V8W 3P6, Canada. Phone: (250) 721-7078. Fax: (250) 721-8882. E-mail: wkay{at}uvic.ca.

Applied and Environmental Microbiology, February 2001, p. 750-759, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.750-759.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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