Comparison of the Membrane-Filtration Fluorescent Antibody Test, the Enzyme-Linked Immunosorbent Assay, and the Polymerase Chain Reaction to Detect Renibacterium Salmoninarum in Salmonid Ovarian Fluid

Abstract

Abstract. Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 ϫ 10 9 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 ϫ 10 4 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.Bacterial kidney disease (BKD), caused by the gram-positive bacterium Renibacterium salmoninarum, affects wild and cultured salmonid stocks worldwide. 13,17,21 The bacterium is unique because not only can it be transmitted horizontally 31 like many other fish pathogenic microorganisms, 40 but also it can be transmitted vertically in association with eggs from infected parents. 8,15 Several mechanisms have been postulated for the egg-associated transmission of R. salmoninarum. 7,23 Many consider vertical transmission to be a consequence of maternal R. salmoninarum infections 16,34 because there is no experimental evidence to date for a contribution by an infected male. 16 As salmonid females reach sexual maturity, R. salmoninarum infections may be found in several organs and body fluids, 33 including the ovarian fluid that surrounds maturing eggs. 22,34 The relative importance of infections in various organs to the success of vertical transmission is poorly understood, but the ovarian fluid can contain more than 1 ϫ 10 9 R. salmoninarum cells/ml, 34 suggesting that this fluid may play a key role in eggassociated transmission of BKD. Whereas the prevalence of R. salmoninarum infections among fish in different progeny groups generally increases as the concentrations of the bacterium in the ovarian fluid be- Received for publication July 14, 1997. come higher, 22,34 evidence exists that vertical transmission of this bacterium may also occur when there are very low numbers of bacterial cells in the ovarian fluid. Renibacterium salmoninarum infections have been detected in chinook salmon (Oncorhynchus tshawytscha) smolts that were the progeny of females with as few as 28 bacterial cells/ml in their ovarian fluid. 22 Broodstock segregation has shown prom...

Detection and Quantification of Renibacterium Salmoninarum DNA in Salmonid Tissues by Real-Time Quantitative Polymerase Chain Reaction Analysis

“…17 Direct comparisons of samples from naturally infected fish by use of ELISA, FAT, and PCR analysis have indicated higher sensitivity of a nested PCR than that of both of the other tests for detection of R. salmoninarum in kidney tissue 4 and coelomic fluid. 15 Although PCR procedures may be more sensitive than ELISA for R. salmoninarum detection, they differ from ELISA in that conventional PCR analyses cannot quantify infection levels and, therefore, cannot be used to determine the severity of infection in R. salmoninarum-positive fish in a population, or to monitor changes in infection levels over time or in response to various treatments. The recent development of real-time quantitative PCR (qPCR) analysis allows the accurate determination of the initial template concentration by monitoring the real-time progress of the PCR assay, 9 and has been used for quantification of target nucleic acid copies.…”

“…22 Several aspects of that procedure differ from the one reported here, but the most notable distinction is the selection of the primers and probes. The primer and probe sequences designed for the recently published qPCR procedure 22 are located on the msa gene between the first-round forward and reverse primer sequences previously selected 15 for the nested PCR procedure that is most widely used for R. salmoninarum detection. 13,25 The primers and probe designed for the qPCR assay described here are unique sequences from a different region of the msa gene, selected to avoid the possibility of contamination from nested PCR amplicons in laboratories that use nested and qPCR procedures for R. salmoninarum detection.…”

“…The DNA was amplified by use of the nested PCR assay as described, 15 with the following modifications: 5 ml of extracted DNA was used as template DNA in the first round of PCR analysis, and 0.4 mM each primer was used in the reaction mixture for the first round of amplification and the nested round of amplification. The first-round PCR assay was performed in a thermal cycler, c with 30 cycles of denaturing at 94uC for 30 seconds, annealing at 60uC for 30 seconds, and extension at 72uC for 1 minute.…”

“…The nested amplification was completed using the same thermal cycler, except that only 20 cycles were performed. The PCR products were analyzed by agarose gel electrophoresis as described, 15 and samples were considered positive if the anticipated 320-bp product was observed.…”

First Record of Renibacterium Salmoninarum in the Sea Lamprey (Petromyzon Marinus)

“…A DNeasy tissue extraction kit (Qiagen, Valencia, California, USA) was used for the extraction of DNA from 100 ml aliquots of kidney tissue homogenates. The DNA was extracted according to the manufacturer's instructions, with a few minor modifications from the method described by Pascho et al (1998). The tissue pellets were obtained by centrifugation at 6000 3 G for 20 min at 4 C, and the pellets were incubated with lysozyme buffer consisting of 180 ml of 20 mg lysozyme (Sigma Chemical, St. Louis, Missouri, USA), 20 mM Tris-HCl, pH 8.0, 2 mM EDTA (Sigma), and 1.2% (v/v) Triton X 100 (Sigma) at 37 C for 1 hr.…”