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Title: TaqMan real-time polymerase chain reaction assay for rapid detection of the fish pathogen Flavobacterium columnare

item Panangala, Victor
item Shoemaker, Craig
item Klesius, Phillip

Submitted to: Aquaculture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2007
Publication Date: 5/15/2007
Citation: Panangala, V.S., Shoemaker, C.A., Klesius, P.H. 2007. TaqMan real-time polymerase chain reaction assay for rapid detection of the fish pathogen Flavobacterium columnare. Aquaculture Research. 38: 508-517.

Interpretive Summary: Flavobacterium columnare ranks as the second most important bacterial fish pathogen in the warmwater aquaculture industry, exceeded in frequency by Edwardsiella ictaluri (the causative agent of enteric septicemia in channel catfish). Columnaris disease caused by the bacterium affects a wide spectrum of both wild and cultured food fish as well as several ornamental fish species world wide. In intensive fish farming operations with high stocking rates and suboptimal environmental conditions, mortality from F. columnare could range between 50 - 60% loss of approximately $50 million annually to fish farmers. Disease management is therefore a priority because even a modest drop in production due to disease could have a devasting impact on the commercial aquaculture industry. Not only do we need to understand the severity of infection but, also the underlying risk to the entire fish population in a pond or farm. Recent innovative developments in polymerase chain reaction (PCR) technology have advanced to the stage where precise information on an infection is generated in quantitative terms. The quantitative TaqMan real-time PCR technique developed by our laboratory is a rapid, specific, sensitive and reproducible assay for detecting the infecting Flavobacterium columnare in diseased fish, as well as determining in quantitative terms the exact amount of pathogen present in an individual fish such as in brood stock, a fish hatchery or among wild fish and in environmental samples where fish live.

Technical Abstract: Flavobacterim columare is a ubiquitous Gram-negative bacterium that causes columnaris disease in a wide variety of fish world wide. Timely detection of disease is important to prevent its spread and reduce the economic loss to fish farmers. We developed a TaqMan-based real-time PCR targeting a 113 bp nucleotide region of the chrondroitin AC lyase gene of F. columnare G4. Specificity of the assay evaluated with 20 isolates of F. columnare and 15 other taxonomically or ecologically related bacteria revealed that that the primers and probe were 100% specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures was observed to be ~3 cells. the lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear (R squared = 0.994) over the range (5.4 ng-5.4fg). In tissues (blood, gills and kidney) of F. columnare infected fish, the bacterial load ranged from 3 to 950,000 CFU per ml. In both, F. columnare infected and spiked samples the positive results were confirmed by bacteriological culture with 100% agreement. The TaqMan real-time PCR developed in the study is specific, sensitive and reproducible for the detection and quantitation of F. columnare in infected fish.