The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum

Abstract

Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response.

Figure 1

Effect of R. salmoninarum challenge on serum nitrate levels in rainbow trout. Fish were injected intraperitoneally with PBS (control) or with one of four strains of R. salmoninarum at 1 × 10⁸ bacteria/fish. Bars represent the mean + SE of 10 fish. *P < 0·05; **P < 0·001.

Figure 2

Effect of R. salmoninarum challenge (1 × 10⁸ bacteria/fish of strain MT 426) on serum nitrate levels in rainbow trout immunized 4 weeks earlier with PBS or killed R. salmoninarum preparations at (a) 1 × 10⁷ bacteria/fish or (b) 1 × 10⁸ bacteria/fish, with or without Freund’s incomplete adjuvant (FIA). All groups were injected i.p. with the live bacteria except for the control fish which were injected with PBS. Bars represent the mean+SE of 10 fish. *P < 0·05.

Figure 3

Detection of iNOS (a) and β‐actin (b) expression in various tissues of rainbow trout 24 hr after i.p. challenge with R. salmoninarum (strain MT 426). Lanes: 1, blood; 2, gill; 3, gut; 4, head kidney; 5, liver; 6, spleen. Data are for one fish and are representative of the three fish analysed.

Figure 4

Time course of iNOS mRNA expression in gill and head kidney of three rainbow trout after i.p. challenge with R. salmoninarum (strain MT 426). Lanes 1, 5 and 9, iNOS transcript in gill; lanes 2, 6 and 10, iNOS transcript in head kidney; lanes 3, 7 and 11, β‐actin transcript in gill; lanes 4, 8 and 12, β‐actin transcript in head kidney. (a) 3 hr after i.p. challenge; (b) 6 hr after i.p. challenge; (c) 12 hr after i.p. challenge.

Figure 5

Time course of iNOS mRNA expression in gill and head kidney of three rainbow trout after i.p. challenge with R. salmoninarum (strain MT 426). Lanes 1, 5 and 9, iNOS transcript in gill; lanes 2, 6 and 10, iNOS transcript in head kidney; lanes 3, 7 and 11, β‐actin transcript in gill; lanes 4, 8 and 12, β‐actin transcript in head kidney. (a) One day after i.p. challenge; (b) 3 days after i.p. challenge; (c) 5 days after i.p. challenge.

Figure 6

iNOS expression in tissues of three rainbow trout (out of six analysed) 24 hr after immersion challenge with R. salmoninarum (strain MT 426). Lanes 1, 7 and 13, iNOS transcript in gill; lanes 2, 8 and 14, iNOS transcript in gut; lanes 3, 9 and 15, iNOS transcript in head kidney. Lanes 4, 10 and 16, β‐actin transcript in gill; lanes 5, 11 and 17, β‐actin transcript in gut; lanes 6, 12 and 18, β‐actin transcript in head kidney.