Molecular diversity and growth features of Flavobacterium columnare strains isolated in Finland

Abstract

Columnaris disease caused by Flavobacterium columnare is a problem in fish farming worldwide. During the last 15 yr, outbreaks have started to emerge in Finland. Flavobacterium columnare Type Strain NCIMB 2248 T and 30 Finnish F. columnare isolates were studied using analysis of 16S rDNA by restriction-fragment length polymorphism (16S RFLP), length heterogeneity analysis of polymerase chain reaction (LH-PCR) products, automated ribosomal intergenic spacer analysis (ARISA), and 16S rDNA sequence analysis. All isolates fell into RFLP Genomovar I and had the same length in the LH-PCR analysis. Based on ARISA, 8 genetically different strains were selected for further analyses. The growth of these strains under different temperatures, NaCl concentrations, and pH values was tested. The Finnish F. columnare strains did not grow at NaCl concentrations >0.1% or at pH values ≤6.5, and they were susceptible to several antimicrobial agents, but not to Polymyxin B or neomycin. These findings may aid in development of methods for disease management at fish farms.KEY WORDS: Flavobacterium columnare · ARISA · RFLP · 16S rRNA gene sequencing Resale or republication not permitted without written consent of the publisherDis Aquat Org 70: [55][56][57][58][59][60][61] 2006 tives for the prevention and treatment of the disease. The use of oxytetracycline is the only method practically applied for the treatment of columnaris disease in Finland, so there is a real threat of development of resistant strains. Therefore, physiological testing and genetic typing of F. columnare isolates is of great importance for understanding the increasing severity of outbreaks and for designing disease management strategies. Tolerance to salinity is of special interest, since columnaris disease has not been reported from coastal areasof Finland, where the salinity of the brackish water is between 2 and 7 ‰. However, F. columnare Type Strain NCIMB 2248 T (National Collection of Industrial, Marine, and Food Bacteria) is known to grow in media with 0.5% NaCl (Bernardet & Grimont 1989).In the present study, we investigated 30 Finnish Flavobacterium columnare isolates obtained from disease outbreaks in northern and central Finland, in order to select representative strains for further analysis. The molecular diversity of these strains was studied using 16S rDNA RFLP, LH-PCR (length heterogeneity analysis of polymerase chain reaction products, Suzuki et al. 1998), and ARISA (automated ribosomal intergenic spacer analysis, Fisher & Triplett 1999). Using this information, 8 genetically different strains were selected for the physiological analysis. The growth patterns of 8 selected strains were studied under different temperatures, salt concentrations, and pH, as well as the susceptibility of the strains to various antibiotics, to gather information for developing potential disease management strategies. MATERIALS AND METHODSBacterial strains and DNA extraction. Thirty Flavobacterium columnare strains isolated from disease outbreaks at Finnish fis...

Intensive fish farming and the evolution of pathogen virulence: the case of columnaris disease in Finland

“…The degradation rate of chondroitin sulphate (ChS) extracted from shark cartilage (Sigma, St Louis, MO, USA) was determined as the change of ChS concentration in relation to total protein concentration of the culture (Suomalainen et al 2006b). The strains were also studied for their population growth characteristics at different temperatures (Suomalainen et al 2006a). A standard volume of bacterial culture cultivated in AO-broth at constant temperature and constant agitation until it reached 50 per cent of the maximum optical density value measured at 595 wavelength (50% saturation) were further cultured at different temperatures on microtitre plates.…”

“…A standard volume of bacterial culture cultivated in AO-broth at constant temperature and constant agitation until it reached 50 per cent of the maximum optical density value measured at 595 wavelength (50% saturation) were further cultured at different temperatures on microtitre plates. Population density measurements were continued until cultures reached 30-50% saturation (Suomalainen et al 2006a). …”

“…Furthermore, the coexistence of several genetically distinct parasite strains in the same population may favour virulence if more virulent strains have a competitive advantage (Nowak & May 1994;Frank 1996;Gandon et al 2001;Read & Taylor 2001), as they do in several systems (Ebert & Mangin 1997;de Roode et al 2005a,b;Bell et al 2006;Ben-Ami et al 2008). Coexistence of genetically different strains of F. columnare has been detected during a disease outbreak at a fish farm (Suomalainen et al 2006a).…”

Intensive aquaculture selects for increased virulence and interference competition in bacteria

“…The MLSA method produces genetic clustering comparable with the ARISA method [25], and the two methods can be used interchangeably. The outlet water strains and one inlet water strain (B355) were genotyped in this study using ARISA, as described earlier [30,32] (see the electronic supplementary material for more details). The Table 1.…”

Phage Specificity of the Freshwater Fish Pathogen Flavobacterium columnare

“…The flavobacterial isolates were subjected to PCR with universal primers (UP-PCR) (for methods, see references 5, 6,, and 13), and the 16S rRNA genes of different groups were sequenced (Table 1). All F. columnare isolates fell into the same UP-PCR group, and thus they were further analyzed with ribosomal intergenic spacer analysis (RISA) (8,29). According to RISA, the isolates were assigned to five groups (Table 1).…”