|Wiens, Gregory - Greg|
Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2012
Publication Date: 12/10/2012
Citation: Marancik, D.P., Wiens, G.D. 2012. A real-time polymerase chain reaction assay for identification and quantification of Flavobacterium psychrophilum and application to disease resistance studies in selectively bred rainbow trout. FEMS Microbiology Letters. doi: 10.1111/1574-6968.12061.
Interpretive Summary: Rapid detection of the bacteria Flavobacterium psychrophilum, which causes bacterial cold water disease (BCWD) in rainbow trout, is crucial to diagnosing and preventing disease in wild and aquaculture-raised fish. We developed a test for the bacteria that allows rapid detection of small amounts of bacteria in fish tissue by recognizing a gene that is only found in F. psychrophilum. This quantitative polymerase chain reaction assay (qPCR) also allows bacterial amounts to be counted. This is useful for comparing infection levels and testing new methods that may reduce bacteria in diseased fish. The assay positively recognized 210 different F. psychrophilum isolates that were collected at fish farms suffering from disease. Twenty-three bacterial species that were not F. psychrophilum were not recognized, which shows that the test is specific for the bacteria of interest. We applied this test to diagnose F. psychrophilum from rainbow trout tissue in naturally occurring BCWD outbreaks and to quantify bacterial loads in experimentally infected rainbow trout. This assay will be applied to disease surveillance practices and future studies to characterize disease in fish selectively bred for BCWD resistance.
Technical Abstract: Rapid detection and quantification of Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) in rainbow trout, is crucial to disease surveillance and encompasses an essential component of BCWD research. Real-time, or quantitative, PCR (qPCR) assays that have previously targeted the 16S rDNA subunit of F. psychrophilum are complicated by polymorphisms and off-target amplification. Insignia primer and probe development software was used to identify a conserved single copy signature sequence in the F. psychrophilum genome that codes for a hypothetical protein with unknown function. Primer and probes were used in a TaqMan qPCR assay that amplified 210 F. psychrophilum isolates with a lower limit of linear detection at 3.1 GE (genome equivalents) per reaction, with no amplification of 23 non-target bacterial isolates. The assay was not inhibited by host spleen DNA or spleen homogenate. Methods were successfully applied to detect F. psychrophilum in rainbow trout from naturally occurring BCWD outbreaks and to quantify bacterial loads in experimentally infected rainbow trout. This assay will be applied to future studies to characterize disease pathogenesis in fish selectively bred for BCWD resistance.