Fig. 5

Gamma irradiation induces reactivation resulting in increased mortality due to uncontrolled growth of mycobacteria.

(A–C) Zebrafish (n = 17) with a latent M. marinum infection were irradiated twice with 25 Gy with one month between the irradiations. Twice irradiated, non-infected zebrafish (n = 23) as well as zebrafish with a latent infection (n = 14) were included as controls. (A) Survival was followed for 30 days after the second dose. *P<0.05. (B) During this period, moribund or recently dead fish were collected 15–22 days after the second radiation dose. Bacterial loads were compared with those of similarly infected, non-irradiated control fish that were collected at the end-point of the experiment. *P<0.05 (C&D) A representative Ziehl-Neelsen stained sample from a reactivated fish showing large numbers of free mycobacteria (purple areas) in the zebrafish body cavity (C). The sides of the body cavity are marked with arrowheads O = ovary, P = pancreas, L = liver, G = gut, K = kidney. (D) A picture taken with a higher magnification showing individual rods (few examples pointed out with arrows). (E) Four groups of 4 adult zebrafish (1 rag2-gfp, 1 lck-gfp and 2 wild-type groups) were γ-irradiated with 25 Gy. Similar control groups were left untreated. Kidneys were collected 8 d post irradiation, pooled and analyzed by FCM. FSC-SSC -plots were gated based on cell size and granularity as described in [56] (gates shown in Figure S3) to assess the effect of irradiation on leukocyte populations. *P<0.05. For further verification of the effect of radiation on lymphocytes, a GFP gate was used for the rag2 and lck groups expressing GFP in B and T cells, or T cells, respectively. (F) Adult non-infected wt zebrafish were irradiated with 25 Gy once (grey bars) (n = 3) or twice (n = 7) (black bars) with one month between the doses. Leukocyte recovery and re-depletion were assessed by FCM. Non-irradiated fish (n = 4) were used as controls. *P<0.05 (G) Fish with a latent infection (n = 7) were irradiated twice with 25 Gy with one month between the doses and plated +/- Rpf for 18 d after the second radiation dose. (H) Fish (n = 6) with a latent infection were plated +/- Rpf.