Carbadox

For the Brazilian isolates the following antibiotics were used: Chlortetracycline hydrochloride, lincomycin hydrochloride, and tylosin tartrate obtained as pure chemicals from Sigma Aldrich (St. Louis, MO, USA). Tiamulin hydrogen fumarate and valnemulin hydrochloride were supplied as pure chemicals from Novartis Animal Health (Switzerland, Basel). For the Thailand isolates, amoxicillin, Zinc-bacitracin, carbadox, enrofloxacin, gentamicin sulfate, polymyxin B (colistin), spectinomycin dihydrochloride, sulfamethazine and trimetroprim were also used and obtained as pure chemicals from Sigma Aldrich (St. Louis, MO, USA). The combination of lincomycin-spectinomycin was prepared as a 1:1 ratio for determination of the combined activity. The working solutions of tested antimicrobials were prepared as previously described [5 (link)]. Briefly, the antimicrobial stock solutions were prepared to a final concentration of 2560 μg/ml and were filtrated through 0.2 μm-pore size filters..A series of two-fold dilutions of the stock solution were made and then further diluted 1:10 with culture medium. The final concentrations of tested antimicrobials were 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 μg/ml. Each concentration of antimicrobial was tested in triplicate and each strain of L. intracellularis was tested twice from two independent bacterial passage.

Cells were treated for 48 h beginning at day 2 of differentiation with DMSO (vehicle control, ATCC), aceclidine (Sigma-Aldrich), acyclovir (Sigma-Aldrich), alloxazine (Sigma-Aldrich), carbadox (Sigma-Aldrich), felodipine (Sigma-Aldrich), GW5074 (Sigma-Aldrich), isoproterenol (Sigma-Aldrich), isradipine (Sigma-Aldrich), lacidipine (Sigma-Aldrich), nafadotride (Tocris Bioscience), nandrolone (Sigma-Aldrich), nifedipine (Sigma-Aldrich), nilvadipine (Sigma-Aldrich), alloxazine (Sigma-Aldrich) diluted in cell-type specific differentiation media at doses listed in figures.