Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.
BenediktsdóttirEHelgasonSGudmundsdóttirS: 1991, Incubation time for the cultivation of Renibacterium salmoninarum from Atlantic salmon, Salmo salar. J Fish Dis14: 97–102.
2.
BenveniskyNReshefL: 1986, Direct introduction of genes into rats and expression of the genes. Proc Natl Acad Sci USA83: 9551–9555.
3.
BrunoDW: 1988, The relationship between auto-agglutination, cell surface hydrophobicity and virulence of the fish pathogen Renibacterium salmoninarum. FEMS (Fed Eur Microbiol Soc) Microbiol Lett51: 135–140.
4.
ChaseDMPaschoRJ: 1998, Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney. Dis Aquat Org34: 223–229.
5.
ElliottDGMcKibbenCL: 1997, Comparison of two fluorescent antibody techniques (FATs) for detection and quantification of Renibacterium salmoninarum in coelomic fluid of spawning chinook salmon. Oncorhynchus tshawytscha. Dis Aquat Org30: 37–43.
6.
ElliottDGPaschoRJ: 2001, Evidence that coded-wire-tagging procedures can enhance transmission of Renibacterium salmoninarum in Chinook salmon Oncorhynchus tshawytscha. J Aquat Anim Health13: 181–193.
7.
EvelynTPT: 1977, An improved growth medium for the kidney disease bacterium and some notes on using the medium. Bull Off Int Epizoot87: 511–513.
8.
EvelynTPT: 1993, Bacterial kidney disease–-BKD. In: Bacterial diseases of fish, ed. InglisVRobertsRJBromageNR, pp. 177–195. Blackwell Scientific Publications, Oxford, UK.
9.
HeidCAStevensJLivakKJWilliamsPM: 1996, Real time quantitative PCR. Genome Res6: 986–994.
10.
LeuteneggerCMKleinDHofmann-LehmannR: 1999, Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan® fluorogenic real-time detection system. J Virol Methods78: 105–116.
11.
McKibbenCMPaschoRJ: 1999, Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tshawytscha. Dis Aquat Org38: 75–79.
12.
MeyersTRThrowerFShortS: 1999, Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan Chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater. Dis Aquat Org35: 101–105.
13.
O'FarrellCLStromMS: 1999, Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene msa in virulent and attenuated strains of Renibacterium salmoninarum. Dis Aquat Org38: 115–123.
14.
OIE (Office International des Epizooties): 2003, Manual of diagnostic tests for aquatic animals, 4th ed., pp. 167–184. OIE, Paris, France.
15.
PaschoRJChaseDMMcKibbenCL: 1998, Comparison of the membrane filtration fluorescent antibody test, enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid ovarian fluid. J Vet Diagn Invest10: 60–66.
16.
PaschoRJElliottDG: 2003, Bacterial kidney disease. In: AFS-FHS (American Fisheries Society–-Fish Health Section) blue book: Suggested procedures for the detection and identification of certain finfish and shellfish pathogens, 2004 ed. AFS-FHS, Bethesda, MD.
17.
PaschoRJElliottDGChaseDM: 2002, Comparison of traditional and molecular methods for detection of Renibacterium salmoninarum. In: Molecular diagnosis of salmonid diseases, ed. CunninghamCO, pp. 157–209. Kluwer Academic Press, Dordrecht, The Netherlands.
18.
PaschoRJElliottDGMallettRWMulcahyD: 1987, Comparison of five techniques for the detection of Renibacterium salmoninarum in adult coho salmon. Trans Am Fish Soc116: 882–890.
19.
PaschoRJElliottDGStreufertJM: 1991, Brood stock segregation of spring Chinook salmon Oncorhynchus tshawytscha by the use of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody technique (FAT) affects the prevalence and levels of Renibacterium salmoninarum in progeny. Dis Aquat Org12: 25–40.
20.
PaschoRJGoodrichTDMcKibbenCL: 1997, Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens. J Aquat Anim Health9: 99–107.
21.
PaschoRJMulcahyD: 1987, Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Can J Fish Aquat Sci44: 183–191.
22.
PowellMOverturfKHoggeCJohnsonK: 2005, Detection of Renibacterium salmoninarum in chinook salmon, Oncorhynchus tshawytscha (Walbaum), using quantitative PCR. J Fish Dis28: 615–622.
23.
RhodesLDCoadyAMDeinhardRK: 2004, Identification of a third msa gene in Renibacterium salmoninarum and the associated virulence phenotype. Appl Environ Microbiol70: 6488–6494.
24.
RockeyDDGilkeyLLWiensGDKaattariSL: 1991, Monoclonal antibody-based analysis of the Renibacterium salmoninarum p57 protein in spawning chinook and coho salmon. J Aquat Anim Health3: 23–30.
25.
USFWS and AFS-FHS (US Fish and Wildlife Service and American Fisheries Society-Fish Health Section): 2004, Standard procedures for aquatic animal health inspections. In: AFS-FHS blue book: Suggested procedures for the detection and identification of certain finfish and shellfish pathogens, 2004 ed. AFS-FHS, Bethesda, MD.
