Location: Warmwater Aquaculture Research UnitTitle: A real time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi Author
Submitted to: Journal of Aquatic Animal Health
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/13/2011
Publication Date: 12/15/2011
Publication URL: http://handle.nal.usda.gov/10113/55863
Citation: Griffin, M.J., Mauel, M.J., Greenway, T.E., Khoo, L.H., Wise, D.J. 2011. A real time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi. Journal of Aquatic Animal Health. 23(4):178-188. Interpretive Summary: The manuscript describes the development of a molecular technique to detect and quantify the numbers of Edwardseilla ictaluri in pond water. This quantitative polymerase chain reaction method will also for better management of enteric septicemia of catfish caused by the organism as it will allow for epidemiological studies, as well as evaluation of vaccination, antibiotic treatments and restrictive feeding practices which are used to manage the disease.
Technical Abstract: A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri–specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.