FIG. 4.

ΔRD1 is attenuated in adult zebrafish and produces a greater proportion of solitary, loosely associated, nonnecrotic granulomas than does infection with WT M. marinum. (A) Survival of adult zebrafish infected with WT or ΔRD1. Fifteen fish were injected with 50 CFU of WT or 1,318 CFU of ΔRD1 bacteria and were reared in separate tanks. Each tank was monitored for mortalities over a 16-week time period. A Kaplan-Meier curve was calculated for each group of fish, and compared to WT-infected fish, ΔRD1-infected fish showed a significant difference in survival as calculated by the log-rank test (P = 0.0012). The Cox proportional hazards model was used to quantify the difference between WT- and ΔRD1-infected fish and confirmed significantly increased survival in the ΔRD1-infected fish, with an HR of 0.1592, a 95% CI of 0.04 to 0.57, and a P value of 0.005. (B) ΔRD1 granulomas are more likely to be solitary, nonnecrotizing, and loosely associated than are WT lesions. A total of 121 WT and 126 ΔRD1 granulomas in infected fish at 16 weeks were scored in a blinded fashion for the presence of necrosis and residence in a multicentric lesion. The subset of nonnecrotic lesions was further assessed for the nature of cellular association (compact or loose). Differences in the proportion of granulomas falling into each group were assessed using contingency tables to calculate RR and 95% CI, assigning ΔRD1 as the experimental group and the wild type as the control group. Significance was determined using Fisher's exact test (**, P < 0.05). Values obtained for nonnecrotic lesions were an RR of 1.850, a 95% CI of 1.394 to 2.456, and a P value of <0.0001; values obtained for solitary lesions were an RR of 1.939, a 95% CI of 1.557 to 2.415, and a P value of <0.0001; values obtained for loose lesions were an RR of 1.337, a 95% CI of 1.041 to 1.718, and a P value of 0.0111. (C and D) Histopathology of adult zebrafish infected for 16 weeks with either 100 CFU of WT (C) or 1,318 CFU of ΔRD1 (D) bacteria. Median bacterial loads at the time of examination were not significantly different (P = 0.17 by Mann-Whitney test). Hematoxylin and eosin staining of pancreatic lesions is shown. Arrows indicate compact, tightly interdigitated epithelioid macrophages in C, while arrowheads in D show loosely associated macrophage aggregates with minimal epithelioid transformation. Scale bars, 50 μm. Magnification, ×100.