|Overturf, Kenneth - Ken|
Submitted to: Journal of Fish Diseases
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/1/2005
Publication Date: 12/1/2005
Citation: Overturf, K.E., Powell, M., Johnson, K., Hogge, C. 2005. Detection of renibacterium salmoninarum in chinook salmon oncorhynchus tshawytscha using quantitative pcr.. Journal of Fish Diseases, 2005, 28, 615-622 Interpretive Summary: Bacterial kidney disease is a systemic infection of salmonids that commonly causes high mortality in wild and aquaculture reared stocks of salmonids. BKD is caused by Renibacterium salmoninarum. Disease outbreaks can range from subacute to chronic cause greater than 50% mortality within an infected population. The problem with the control of this disease is that fish can carry and shed the bacteria for years and show no signs of disease. It can apparently survive within the macrophage and not kill or be killed. At the Hagerman Fish Culture Experiment Station in Hagerman, Idaho we have developed a molecular assay using real-time polymerase chain reaction and fluorescent labeled probes for the detection and quantification of this bacterial organism. This method is much more rapid than the old method of using enzyme-linked immunosorbent assays and polyclonal antibodies, also at low levels of the organism the method is more highly sensitive and accurate in quantifying the presence and number of organisms within the sample.
Technical Abstract: We have developed a quantitative PCR assay to detect varying levels of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). This assay allows for the direct enumeration of bacterial DNA or RNA copy number within tissues and body fluids. The assay can be applied nonlethally and can be used to determine whether R. Salmoninarum is transcriptionally acive. The presence of R. salmoninarum in kidey tissues from 430 chinook salmon collected from 5 Idaho Fish and Game operated hatcheries was initially evaluated using the widely employed enzyme-linked immunosorbent assay (ELISA) with two sets of Kirkegaard and Perry Laboratories (KPL) polyclonal antibodies, "mother batches" 1 & 2. The same tissue samples were then analyzed using the novel quantitative PCR assay and the results compared. At moderate to high levels of infection (O.D. values >0.5) ELISA and estimated DNA copy numbers were highly correlated (r2>0.80) though correlation to specific antibody batches varied. However, lower ELISA values (O.D. <0.5) observed with either antibody batches did not correlate well with the quantitative PCR assay (r2<0.43). Negative controls run concurrently with the PCR assay did not indicate extraneous DNA contamination in the PCR samples.