Ecological changes affect pathogen epidemiology and evolution and may trigger the emergence of novel diseases. Aquaculture radically alters the ecology of fish and their pathogens. Here we show an increase in the occurrence of the bacterial fish disease Flavobacterium columnare in salmon fingerlings at a fish farm in northern Finland over 23 years. We hypothesize that this emergence was owing to evolutionary changes in bacterial virulence. We base this argument on several observations. First, the emergence was associated with increased severity of symptoms. Second, F. columnare strains vary in virulence, with more lethal strains inducing more severe symptoms prior to death. Third, more virulent strains have greater infectivity, higher tissue-degrading capacity and higher growth rates. Fourth, pathogen strains co-occur, so that strains compete. Fifth, F. columnare can transmit efficiently from dead fish, and maintain infectivity in sterilized water for months, strongly reducing the fitness cost of host death likely experienced by the pathogen in nature. Moreover, this saprophytic infectiousness means that chemotherapy strongly select for strains that rapidly kill their hosts: dead fish remain infectious; treated fish do not. Finally, high stocking densities of homogeneous subsets of fish greatly enhance transmission opportunities. We suggest that fish farms provide an environment that promotes the circulation of more virulent strains of F. columnare. This effect is intensified by the recent increases in summer water temperature. More generally, we predict that intensive fish farming will lead to the evolution of more virulent pathogens.

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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...

Fish farming creates conditions where disease transmission is enhanced and antibiotic treatments are commonly used to cure bacterial diseases to prevent severe losses due to infections. Ability to persist in such an environment has been suggested to lead to the evolution of high virulence. Columnaris disease caused by Flavobacterium columnare is a growing problem in freshwater fish farming. Transmission of the disease is poorly known, and survival of F. columnare in the rearing environment has not been studied. This paper addresses both transmission of columnaris disease and survival strategy of F. columnare. Saprophytic activity of F. columnare was studied by infecting rainbow trout fingerlings before and immediately after death and by following bacterial shedding from the fish carcasses. From fish killed immediately after infection, bacteria were shed at high rates for 5 days, and from fish exposed to F. columnare post mortem for 8 days. In another experiment, rainbow trout fingerlings were experimentally infected with F. columnare and monitored for transmission of the bacteria post infection until and after the death of the fish. The transmission of columnaris disease to living rainbow trout was the most efficient from dead fish, from which bacteria were shed into water at higher rates than from living fish. We also found that F. columnare can survive at least for 5 months in both sterilized distilled and lake water. These results show that death of the host causes no cost for F. columnare; it thrives in alive and dead fish, and in water. Saprophytism may have been a transition stage to pathogenicity of this originally harmless water bacterium, and maintained as an effective transmission and survival strategy of F. columnare. Our findings also suggest that F. columnare may be able to persist in the rearing environment during antibiotic treatments of the living fish.