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Diseases of Aquatic Organisms

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DAO 51:93-100 (2002)  -  doi:10.3354/dao051093

Diagnosis of flavobacteriosis by direct amplification of rRNA genes

Marja Tiirola1,*, E. Tellervo Valtonen1, Päivi Rintamäki-Kinnunen2, Markku S. Kulomaa1

1Department of Biological and Environmental Science, University of Jyväskylä, PO Box 35 (YAB), 40351 Jyväskylä, Finland
2Department of Biology, University of Oulu, 90014 Oulu, Finland

ABSTRACT: A broad-range bacterial PCR method with universal 16S rDNA targeting primers and bacterial cultivation was used to identify the putative pathogen in flavobacterial outbreaks. Restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the partial 16S rDNA PCR products of 10 skin samples and 10 representative isolates derived from the same fish specimens revealed differences between direct molecular and cultivation-based analysis. Flavobacterium columnare-like sequences dominated in the direct molecular analysis in most cases, whereas most of the isolates belonged to a phylogenetically heterogeneous group of flavobacteria clustering with F. hibernum. F. columnare was isolated in only 1 outbreak. The possible explanations for the different results may be attributable to difficulties in the plate cultivation procedure of external flavobacterial samples. During plate cultivation, the dominating Flavobacterium species can be masked by saprophytic species of the same genus or other genera, or the growth of flavobacteria can be completely inhibited by antagonistic bacteria such as Pseudomonas. Direct analysis of the prevailing 16S rDNA sequences avoids the problems with cultivation and may thus be preferable for the diagnosis of flavobacterial diseases. When isolating flavobacteria from external samples, serial dilution of the sample before plating can improve the results.

KEY WORDS: Broad-range PCR amplification · Flavobacterium · Identification · Antagonism

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