Bacterial kidney disease (BKD) has been a significant problem for over 50 years in the Great Lakes basin (GLB) where it has been associated with large-scale fish mortalities and chronic infections in feral and propagated fish. The most commonly used and widely accepted method for detection of R. salmoninarum is a sandwich enzyme-linked immunosorbent assay (ELISA) on lethally collected kidney and spleen samples. While ELISA is relatively fast and more efficient than other serological assays, there are some concerns regarding what could be overlooked as a result of conducting a single assay on specific samples. Herein, I describe several factors that could affect the detection of BKD in feral and experimentally infected fish that are often overlooked upon assay or sample choices. Using data from the past decade, a decline in the overall presence of BKD in feral and hatchery-raised Chinook salmon (Oncorhynchus tshawytscha) (CHS), coho salmon (O. kisutch), and steelhead (O. mykiss) was observed. This also coincided with the implementation of enhanced biosecurity measures at state hatcheries and gamete-collection. Moreover, to potentially reduce the dependency on lethally collected samples for regular screening, the detection of R. salmoninarum in other sample types, such as mucus, blood, and a urine/feces mixture, was evaluated. All sample types were collected from experimentally infected CHS and compared for their efficiency to detect the presence of R. salmoninarum by standard bacterial culture techniques, nested polymerase chain reaction (nPCR), and sandwich ELISA. It was found that the urine/feces mixture was the preferred non-lethal sample, and that a combination of assays and samples greatly increased the likelihood of detecting R. salmoninarum. Not only does collecting several samples enhance the detection of R. salmoninarum, but the presence of R. salmoninarum in the urine/feces mixture suggests that the bacterium may utilize the anal opening as a portal of entry. To determine if anti-R. salmoninarum antibodies are elicited during an infection, and to establish a protocol for their detection, a single-dilution indirect-ELISA was modified and assessed for its usefulness in detecting and semi-quantifying levels of elicited antibodies. The ability of the indirect-ELISA to detect antibodies was evaluated in several groups of experimentally infected rainbow trout and feral Oncorhynchus spp. The antibody response observed in the experimentally infected fish suggested their response was relatively short-lived. On the contrary, data from the spawning adult fish provided evidence of elevated antibodies, implying that they have been exposed multiple times to R. salmoninarum while existing in the GLB. A thorough analysis of R. salmoninarum infections was also conducted on over 600 CHS returning to spawn at several GLB gamete-collecting weirs to assess infection levels, shedding, and potential disease progression. Results suggested that female fish could be more susceptible to R. salmoninarum infection, but males may still play a role in shedding of the bacterium. Low levels of circulating anti-R. salmoninarum antibodies observed in the late spawning CHS, along with high intensities of infection, are evidence that fish returning to spawn later in the season are more heavily infected with R. salmoninarum than those early in the season. To this end, the detection of R. salmoninarum appears to depend upon several factors (i.e., type of sample and assay, time of collection, and fish age) that should be taken into consideration before deciding how to diagnose BKD in a particular fish population.
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