The role of reactive oxygen and nitrogen species in the immune response of rainbow trout to Renibacterium salmoninarum
The role of reactive oxygen and nitrogen intermediates in the immune response of rainbow trout to R.s. was investigated. The early events occurring when the pathogen interacted with trout macrophages were assessed in terms of the respiratory burst elicited. Live R.s. elicited a respiratory burst, which was enhanced by heat-killed microorganism. This phenomenon, though, was not observed using UV-killed bacteria. Both responses were enhanced when a combination of LPS and TNF was used to activate the macrophages prior to contact R.s. Further studies demonstrated that both compounds synergised to enhance superoxide (O2) production, and that this was correlated with the ability to kill the pathogen. Opsonisation of R.s. with serum factors also increased the respiratory burst, but no difference was found between normal serum and heat-inactivated serum. The role of NO in the immune response of rainbow trout is also studied. Though no evidence of NO production was found in vitro, i.p. injection of live R.s. produced higher NO levels in serum as compared to controls. Fish injected with a virulent strain showed higher levels of NO than controls and than fish injected with an avirulent strain and other strains of unknown virulence. Fish vaccinated with killed R.s. and FIA also showed a significant increase in NO levels, but only four days after vaccination, decreasing thereafter, at both doses of vaccine tested. Injected of Brivax II, an attenuated strain of Aeromonas salmonicida, did not produce a significant increase of NO. RT-PCR was used to detect the expression of the iNOS in different tissues of rainbow trout. iNOS expression was seen only in gill and kidney after i.p. injection. iNOS was detected in the gills 6 h after injecting live R.s. and the expression was still present at day 5. iNOS was detected in the kidney 24 h after injection but was switched off at day 3. After bath challenge with the bacterium, iNOS was expressed in gill, gut and kidney, but the expression varied in each fish. No iNOS expression was found in macrophages isolated from challenged fish.