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...

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil‐based adjuvant. We evaluated the relative abilities of the bacterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty‐one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 × 103, 1.7 × 105, or 5.3 × 106 live R. salmoninarum cells/mL. An enzyme‐linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney–spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of bacterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either L7 × 104 or l.7 × 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.