Objective Renibacterium salmoninarum is a pathogenic gram‐positive bacterium and is the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK‐90 and ATCC 33739) in Rainbow Trout Oncorhynchus mykiss. Methods The CK‐90 strain was intracoelomically injected (100 μL) at a high dose containing 4.80 × 106 CFU/g of fish (optical density at 525 nm [OD525] = 1.779) and a low dose containing 6.86 × 105 CFU/g of fish (OD525 = 1.077); alternatively, fish were immersed in a solution containing 4.5 × 107 CFU/mL of fish (OD525 = 0.886). The ATCC 33739 strain (originating from Brook Trout Salvelinus fontinalis) was also included and intracoelomically injected at 3.58 × 105 CFU/g of fish (OD525 = 1.431) to discern differences in virulence between the strains. Result Clinical signs of BKD manifested at approximately 10 d postchallenge, and mortalities began at 19 days postchallenge. To confirm infection and quantify R. salmoninarum antigen load, an enzyme‐linked immunosorbent assay (ELISA) was conducted using kidney tissue collected after the challenge. Rainbow Trout that were challenged with CK‐90 by injection (both high‐ and low‐dose groups) exhibited significantly higher mortality than fish that were injected with ATCC 33739 or those that were exposed to CK‐90 via immersion challenge. The R. salmoninarum p57 (57‐kDa protein) antigen was confirmed via ELISA. Antigen load for fish injected with CK‐90 (high dose: OD405 = 0.71; low dose: OD405 = 0.66) was significantly higher than that for fish injected with ATCC 33739 (OD405 = 0.34). The CK‐90 strain (both high and low doses) was more virulent than ATCC 33739, which caused no mortalities over the 28‐days trial. Although there were no mortalities among ATCC 33739 fish, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in those fish. Conclusion The immersion challenge methodology for R. salmoninarum CK‐90 was ineffective for inducing mortalities at the examined dose.

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