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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...

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ABSTRACT. Bacterial kidney disease of salmonid fishes, caused by Renibactenum salrnoninarum, was first reported more than 50 yr ago; nevertheless, large gaps persist in our knowledge of the infectionparticularly in methods for its control. In the 1950's, principal control measures consisted of prophylactic or therapeutic feeding of sulfonamides, which were later supplanted by the antibiotic erythromycin. Chemotherapy has effected some reduction of mortality, but benefits are typically transient and mortality usually resumes after the drug is withdrawn. Some studies have indicated that diet composition affects the prevalence and severity of the disease. Although tests of chemotherapeutants and diet modification have continued, research emphasis has shifted partly toward prevention of the disease by breaking the infection cycle. It is now generally accepted that R. salrnoninarum can be transmitted both vertically and horizontally. Experimental ev~dence indicates that immersion of newly fertilized eggs in iodophor or erythromycin does not prevent vertical transmission. However, the injection of female salmon with erythromycin before they spawn shows promise as a practical means of interrupting vertical transmission. The results of attempts to prevent infection of juvenile salmonids by vaccination against bacterial kidney disease have been disappointing, thus underscoring a basic need for a better understanding of protective mechanisms in salmonids. The recent development of more sensitive and quantitative detection methods should aid in evaluating the efficacy of current and future control strategies.

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

Abstract. 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.

Nucleic acid-based assays have shown promise for diagnosing Renibacter~um salmoninarum in tissues and body fluids of salmonids. Development of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. sahoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. sdmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specdlcity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positi.ve reactions in the enzymelinked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarurn. Kidney samples from 74 naturally infected c h o o k salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43 %, respectively.