Figure 1.
Life expectancy and colonization of zebrafish swimming larvae infected by different pathogenic bacteria.
A. Life expectancy of axenic zebrafish larvae exposed by bath at 6 dpf to E. coli, E. ictaluri or other pathogenic bacteria. Mean survival is represented by a large hyphen. Standard deviations are also indicated. Asterisks indicate significant difference from non-infected population (*p<0.05, **p<0.01, ***p<0.001). B. Colonization of zebrafish larvae infected by different pathogenic bacteria. CFU counts of axenic zebrafish larvae exposed by bath at 6 dpf to E. ictaluri and other pathogenic bacteria. Mean and standard deviations are indicated. (n = 5). C. Colonization of zebrafish gut monitored at 9 dpf ( = 3 days post infection) by transmitted light microscopy (left) and whole-mount immunohistochemistry using a polyclonal antibody recognizing Gram-negative bacteria (right). Arrows indicate bacterial localization within the gut.
Figure 2.
Characterization of zebrafish larva infection by E. ictaluri.
6 dpf germ free or conventional zebrafish larvae exposed to E. coli MG1655 or E. ictaluri by bath immersion were transferred after 6 h to clear autoclaved water. A. Influence of the amount of E. ictaluri in immersion bath on larvae mortality rate monitored daily and presented as in Figure 1. Control populations are shown in gray. B. E. ictaluri CFU number recovered from zebrafish larvae at different days post-infection (Mean ± SD; n = 5). C. E. ictaluri CFU number recovered from zebrafish larvae at 3 days post-infection when infected with different doses of E. ictaluri (Mean ± SD; n = 4). D. E. ictaluri-induced mortality of axenic and conventional zebrafish larvae monitored and presented as in panel A. E. E. ictaluri-induced mortality of axenic and conventional zebrafish larvae as monitored and presented as in panel A.
Figure 3.
Localization of E. ictaluri in infected gnotobiotic zebrafish larvae.
At 3 days post-infection ( = 9 dpf), germ-free zebrafish larvae exposed at 6 dpf to E. ictaluri were analyzed by whole-mount immunofluorescence. Germ-free 9 dpf zebrafish larvae were used as control. A. Localization of E. ictaluri in infected larvae. E. ictaluri cells (red) were detected with a stereomicroscope by immunofluorescence using a polyclonal antibody recognizing Gram-negative bacteria, including E. ictaluri. White arrows pinpoint E. ictaluri main infection sites on zebrafish head and gut. Yellow arrows pinpoint non-specific labeling. B. Details of E. ictaluri insertion in larval intestinal tissue. Clusters of E. ictaluri cells (shown by large white arrows) were observed by confocal microscopy outside the gut lumen, surrounded by zebrafish intestinal cells (nuclei stained in blue). Left panel: 10× objective, transmitted light and red (bacteria) fluorescence overlay; central panel: 40× objective, transmitted light, dashed red lines indicate gut lumen boundaries; right panel: 40× objective, red (bacteria) and blue (nuclei) fluorescence overlay. C. 10× objective. Confocal fluorescence picture of larval head infected by E. ictaluri (red). Zebrafish cell nuclei are shown in blue (DAPI staining). D. Analysis of larval rostrum by fluorescence and Nomarski optics. White arrow shows a bacterial abscess within the oral cavity, whereas the yellow arrow pinpoints E. ictaluri clusters co-localized with external skin lesions. White bars = 50 µm.
Figure 4.
Characterization of the gnotobiotic zebrafish larva immune response to E. ictaluri infection.
Kinetics of inflammation marker expression in zebrafish larvae. qRT-PCR was performed using primers specific to il1b (A), tnfa (B), il22 (C) and il10 (D) (inflammation markers) on RNA extracted from pools of germ-free zebrafish larvae or larvae exposed to E. coli MG1655 (control) or E. ictaluri at 1, 2, and 3 days post-infection or heat-killed 3 dpi E. ictaluri. Levels were standardized to levels of uninfected axenic fish, presented results are mean±SEMof three biological replicates. Asterisks indicate significant difference determined by two-way ANOVA with Bonferroni correction (*p<0.05, **p<0.01, ***p<0.001). E. Localization of il1b expression in zebrafish larvae performed by in situ hybridization on whole-mounted zebrafish larvae treated at 3 dpi.
Figure 5.
Protective effect of selected strains against gnotobiotic zebrafish larva infection by E. ictaluri.
A. Four dpf-old freshly hatched axenic zebrafish larvae were kept germ-free or incubated with selected protective bacterial strains for 2 days prior to exposure to E. ictaluri at 6 dpf. Mortality was monitored daily after E. ictaluri infection. Control non-infected larvae are shown in gray. Asterisks indicate significant difference from E. ictaluri-infected population (***p<0.001). B. Inflammation marker expression in pretreated E. ictaluri-infected larvae. qRT-PCR was performed on 5 individual larvae per group using primers specific to il1b, tnfa and il10 on RNA extracted at 3 dpi ( = 9 dpf) from germ-free or E. coli MG6155 F′ pre-colonized zebrafish larvae exposed to E. ictaluri at 3 days post infection. Results were normalized to mean expression in germ-free animals; mean ± SEM. Asterisk indicates significant difference between E. ictaluri-infected and uninfected populations (**p<0.01, ***p<0.001).
Table 1.
Strains and plasmids used in this study.
Figure 6.
Comparative analysis of neutrophil distribution in pretreated larvae infected or not by E. ictaluri.
The distribution of neutrophils after E. ictaluri infection was examined at 9 dpf in mpx:GFP larvae stained with an anti-GFP antibody (n = 5 to 10 per condition) (A) Representative pictures from which neutrophils were manually counted are shown. Total neutrophils consist of all visible neutrophils except those in the kidney, which is too deep and too dense for cells to be accurately counted. Among these, three subpopulations were counted: head (yellow dots), hematopoietic sites in the trunk and tail (white dots) and gut (red dots). (B). Neutrophil counts; statistical significance was calculated between the corresponding pretreated larvae infected and non-infected by E. ictaluri. (*p<0.05, **p<0.01, ***p<0.001).
Figure 7.
Impact of E. coli adhesive properties on its protective effect in E. ictaluri-infected zebrafish larvae.
A. Effect of introduction of conjugative F plasmid in other E. coli. Four dpf-old freshly hatched larvae were kept germ-free or incubated with E. coli MG1655 or E. coli ED1a-sm with and without the F plasmid for 2 days prior to exposure to E. ictaluri at 6 dpf. Mortality was monitored daily after E. ictaluri infection. Controls are shown in gray (***p<0.001). B. Quantification of E. coli expressing different adhesion factors associated with gnotobiotic zebrafish larvae at 9 dpf. Means and standard deviations of the number of CFU recovered from larvae are reported (n = 4; ***p<0.001). Abbreviations used: F+ for F plasmid; ag43+ for antigen 43; fim for type 1 fimbriae; WT for wild type. C. Mortality rate of larvae pretreated with E. coli derivative strains constitutively expressing different adhesion factors and infected or not (control –gray-) with E. ictaluri (***p<0.001).