title: Real-time polymerase chain reaction assays for the detection and quantification of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella piscicida-like species in catfish tissues and pond water.
abstract: Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida-like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published primers, for E. tarda, E. piscicida, and E. piscicida-like sp. to provide rapid quantitative confirmatory tests for these phenotypically ambiguous bacteria. The qPCR assays were shown to be repeatable and reproducible, with high degrees of sensitivity and specificity. Each assay showed a linear dynamic range covering 8 orders of magnitude and a sensitivity limit of 5 copies of target DNA in a 15-µL reaction. In addition, each assay was found specific to their respective targets with no observed amplification from nontarget organisms, including the closely related E. ictaluri and E. hoshinae. Under the conditions used in this study, the 3 assays had a quantifiable limit ranging from 10(3) (E. piscicida) to 10(2) (E. piscicida-like and E. tarda) colony forming units in kidney tissue biopsies (approximately 25 mg), pond water samples (35 mL), and broth culture (20 μL). In experimental challenges, the assays were able to detect their respective targets in both clinically and subclinically infected channel catfish (Ictalurus punctatus) fingerlings. In addition to quantifying target bacteria from various substrates, the assays provide rapid identification, differentiation, and confirmation of the phenotypically indistinguishable E. tarda, E. piscicida, and E. piscicida-like sp., a valuable tool for diagnostic assessments.
authors.affiliation: Thad Cochran National Warmwater Aquaculture Center, Aquatic Research and Diagnostic Laboratory, College of Veterinary Medicine (Reichley, Ware, Griffin), Mississippi State University, Stoneville, MSThad Cochran National Warmwater Aquaculture Center, Mississippi Agriculture and Forestry Experiment Station (Greenway and Wise), Mississippi State University, Stoneville, MS.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Reichley
authors.affiliation: Thad Cochran National Warmwater Aquaculture Center, Aquatic Research and Diagnostic Laboratory, College of Veterinary Medicine (Reichley, Ware, Griffin), Mississippi State University, Stoneville, MSThad Cochran National Warmwater Aquaculture Center, Mississippi Agriculture and Forestry Experiment Station (Greenway and Wise), Mississippi State University, Stoneville, MS.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Ware
authors.affiliation: Thad Cochran National Warmwater Aquaculture Center, Aquatic Research and Diagnostic Laboratory, College of Veterinary Medicine (Reichley, Ware, Griffin), Mississippi State University, Stoneville, MSThad Cochran National Warmwater Aquaculture Center, Mississippi Agriculture and Forestry Experiment Station (Greenway and Wise), Mississippi State University, Stoneville, MS.
authors.forename: Terrence E
authors.initials: TE
authors.lastname: Greenway
authors.affiliation: Thad Cochran National Warmwater Aquaculture Center, Aquatic Research and Diagnostic Laboratory, College of Veterinary Medicine (Reichley, Ware, Griffin), Mississippi State University, Stoneville, MSThad Cochran National Warmwater Aquaculture Center, Mississippi Agriculture and Forestry Experiment Station (Greenway and Wise), Mississippi State University, Stoneville, MS.
authors.forename: David J
authors.initials: DJ
authors.lastname: Wise
authors.affiliation: Thad Cochran National Warmwater Aquaculture Center, Aquatic Research and Diagnostic Laboratory, College of Veterinary Medicine (Reichley, Ware, Griffin), Mississippi State University, Stoneville, MSThad Cochran National Warmwater Aquaculture Center, Mississippi Agriculture and Forestry Experiment Station (Greenway and Wise), Mississippi State University, Stoneville, MS matt.griffin@msstate.edu.
authors.forename: Matt J
authors.initials: MJ
authors.lastname: Griffin
mesh_terms: D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D020594:Edwardsiella / Q000235:genetics / Q000302:isolation & purification*; D020609:Edwardsiella tarda / Q000235:genetics / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology / Q000662:veterinary*; D005393:Fish Diseases / Q000382:microbiology*; D007059:Ictaluridae; D061005:Ponds; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Edwardsiella piscicida; Edwardsiella piscicida–like; Edwardsiella tarda; real-time polymerase chain reaction