M. marinum infections

Fish were infected either with a low (~40 cfu) or high (~10,000 cfu) dose of M. marinum four weeks after immunization. M. marinum ATCC 927 was cultured at 29°C in standard mycobacterium medium, 7H9 (BD Biosciences), and prepared for infections as described in [8]. For infections, fish were anesthetized with 0.02% 3-aminobenzoic acid ethyl ester. The desired dose of M. marinum diluted in 0.2 M sterile KCl was injected intraperitoneally (i.p.). Thereafter, the fish were immediately released into a recovery tank. Infection doses were verified by plating the bacteria onto 7H10 plates (BD Biosciences). Following the infections, the well-being of the fish was monitored daily, and fish showing signs of stress or mycobacterial disease during the experiment follow-up period were euthanized with 0.04% 3-aminobenzoic acid ethyl ester.