Stress proteins, phagocytes, and pathology in coho salmon with bacterial kidney disease

Bacterial kidney disease (BKD) is an important disease of wild and cultivated salmonid fish in Canada and around the world. It is known to be caused by a slow growing Gram positive bacterium, Renibacterium salmoninarum, which grows as an intracellular pathogen of phagocytic cells. The disease can not currently be prevented and BKD cannot be cured with antibiotic treatment. Stress is a major factor in the susceptibility of fish to BKD and the disease can itself be considered a chronic stressor. The pathology of the disease involves a chronic and progressive spread of the pathogen throughout the organs with an accompanying chronic inflammation and accumulation of fish leukocytes at the site of infection. The inflammatory response and the phagocytes present at the sites of infection are not able to eliminate the pathogen but are thought to contribute to the tissue damage caused by BKD. Stress proteins (SP) are a set of proteins synthesized by cells in vitro and in vivo as a result of exposure of the cells to stressors, such as toxic substances or heat. They can be used to monitor the effects of a variety of stressors. Up to now most of the research on SP has been focussed on induction of SP by physical or chemical stressors which act directly on the cells or tissues in culture. The fact that BKD can act as a chronic stressor upon the tissues of infected fish makes it an attractive system to study the induction of SP in whole animals by physiological processes such as tissue damage caused by phagocytes and the oxidants they produce. I am the first to report SP induction in a fish disease and this work is one of only two known examples of SP induction in fish during pathophysiological processes (Forsyth et al, 1997; Janz et al, 1997). Using an enzyme linked immunosorbent assay (ELISA) for HSP-70 and immunofluorescent staining of tissue sections, I found that in the tissue of coho salmon (Oncorhynchus kisutch) with active BKD, host HSP-70 was induced in the vicinity of bacterial microcolonies. This localized SP expression may have important implications for the pathology of BKD and possibly for diseases caused by other Gram positive bacteria and intracellular pathogens. The presence of elevated HSP-70 levels in these diseased fish also raises the practical concern that disease in natural populations might interfere with the use of SP levels as an indicator of environmental stress. Investigators studying SP expression should be careful to examine the sampled animals for signs of infectious disease as a potential confounding factor. I found no compelling evidence to support the hypothesis that this SP induction resulted from the action of phagocytes at the sites of infection. Culture experiments with rainbow trout (Oncorhynchus mykiss) phagocytes, with hepatocytes and with mixtures of those cells support the idea that salmonid phagocytes may have a weak stress response, at least when cultured in vitro. The hepatocyte cultures had a strong SP response to heat shock, but in mixed cultures I saw no evidence for SP induction by phagocyte oxidants or direct induction by R. salmoninarum. I conclude that other less obvious mechanisms should be considered and that the process of SP induction in BKD may be more complicated than can easily be modeled in a simple culture system. I suggest some possible improvements to in vitro and in vivo culture systems to facilitate the study of the interactions between the bacteria and the host cells at sites of infection in fish with BKD. In this course of the experiments with BKD in coho salmon I also made two incidental observations which may be important. The first is that crowding may be useful in challenge experiments as a means to reduce the variability of fish in their susceptibility to BKD. The second is that R. salmoninarum might grow in two different modes, and that the bacterium may switch to a more virulent form when a sufficient density is reached; a quorum sensing system may control virulence in R. salmoninarum.Land and Food Systems, Faculty ofGraduat