Lipoclodronate macrophage depletion results in lethal M. fortuitum infection. (A) At 24 h post-fertilization, embryos were treated with liposomal clodronate via caudal vein injection to transiently deplete macrophages. At 30 h post-fertilization, embryos were injected intravenously with 300 CFU of GFP-expressing M. fortuitum with embryo survival monitored over a 12 day period. n = 20–25 embryos/group. Statistical analysis was completed using the log-rank (Mantel-Cox) statistical test for survival curves. Data shown is the merge of two independent experiments. (B) Bacterial burden at 2 days post-infection was calculated using fluorescent pixel count (FPC) determination with ImageJ software. Lipoclodronate-treated embryos were normalized against corresponding controls in each experiment. Each datapoint represents an individual embryo. Error bars represent standard deviation. Statistical significance was determined by Student′s t-test. Plots represent a pool of 2 independent experiments containing approximately 20 embryos per group. (C) Representative images of wild-type (WT) and Lipoclodronate-treated embryos infected with 300 CFU of GFP-expressing M. fortuitum at 2 days post-infection. Scale bars represent 1 mm. (D) The proportion of embryos with bacterial cords and the total distribution of cords categorized as low (<5 cords/embryo), moderate (6–10 cords/embryo) and high (>10 cords/embryo) in M. fortuitum-infected embryo at 2 days post-infection. Error bars represent standard deviation. Data shown is the merge of two independent experiments. Statistical significance was determined by Student′s t-test. (E) The proportion of embryos with abscesses and the average number of abscesses per infected embryo at 2 days post-infection. Error bars represent standard deviation. Data shown is the merge of two independent experiments. Statistical significance was determined by Student′s t-test. **P ≤ 0.01, ****P ≤ 0.0001.
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