|Van santan, V.|
Submitted to: Diseases of Aquatic Organisms
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/29/2006
Publication Date: 3/15/2007
Citation: Panangala, V.S., Shoemaker, C.A., Van Santan, V.L., Dybvig, K., Klesius, P.H. 2007. Multiplex-PCR for simultaneous detection of three fish-pathogenic bacteria, Edwardsiella ictaluri, Flavobacterium columnare and Aeromonas hydrophila. Diseases of Aquatic Organisms 74: 199-208. Interpretive Summary: Edwardsiella ictaluri, Flavobacterium columnare and Aeromonas hydrophila are three major bacterial pathogens of fish that cause a significant economic impact on the aquaculture industry in the USA and world wide. Enteric septicemia of catfish (ESC) caused by E. ictaluri, reported by 67% of operations within one year and columnaris disease caused by F. columnare, reported by 50% of operations within one year, are two of the most devastating bacterial infections accounting for a loss of over fifty million dollars annually to the US catfish farming industry. The three organisms are ubiquitous in the aquatic environment and since fish are commercially reared in extensive pond acreages with high stocking densities (8,000-12,000 fish/pond-acre), the occurrence of concurrent or multiple infections in the same host could not be overlooked. Traditional bacteriological methods for disease diagnosis using culture techniques are highly inefficient to arrive at a quick diagnosis, resulting in increasing the potential for spreading disease and delaying implementation of important control measures. When multiple bacterial pathogens are likely to occur as in the aquatic environment, selective and accurate amplification of multiple target genes in a single reaction is made possible rapidly, by the multiplex-Polymerase chain reaction (m-PCR) developed in our research.
Technical Abstract: A multiplex PCR (m-PCR) method was developed for simultaneous detection of three important fish pathogens in warm water aquaculture. The m-PCR was optimized with appropriate adjustment of reaction buffers and a touchdown protocol to amplify target DNA fragments from Flavobacterium columnare (504-bp), Edwardsiella ictaluri (407-bp) and Aeromonas hydrophila (209-bp). The detection limit was 20 pg of genomic DNA per m-PCR reaction mixture equivalent to 1.5 × 10 to the second colony-forming units (CFU) for F. columnare, 1.6 × 10 to the third CFU for E. ictaluri and 2.3 × 10 to the third CFU for A. hydrophila. The specificity of the m-PCR was evaluated with 10 representative isolates of each of the three bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. Blood, gills and kidney tissues from channel catfish (Ictalurus punctatus Rafinesque) infected with F. columnare, E. ictaluri, and/or A. hydrophila alone or different combinations, and tissues (blood, gills and kidney) from naïve fish spiked with bacteria, yielded amplicons congruous with the infecting organism. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of F. columnare, E. ictaluri and A. hydrophila in infected fish compared to the traditional bacteriological culture techniques that are time consuming.