Sonication does not affect binding and internalization of <i>C. difficile</i> spores by Raw 264.7 cells.

<p>Monolayers of Raw 264.7 cells were infected with untreated (white bars) and sonicated (grey bars) <i>C. difficile</i> spores of strains 630 and Pitt177 at an MOI of 10 for 30 min, and analyzed by fluorescence microscopy for: relative percentage of Raw 264.7 cells with at least one spore (A), relative number of spores per Raw-spore complex (B), and relative percentage of internalization (C) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043635#s2" target="_blank">Methods</a> section and in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043635#pone-0043635-g002" target="_blank">figure 2</a>. Results are the average of at least three independent experiments and error bars are standard error of the mean.</p

Representative fluorescence micrographs of internalization of <i>C. difficile</i> spores by Raw 264.7 cells.

<p><i>C. difficile</i> spores were labeled with biotoin and Alexa Fluor 488 (green) prior to infection of monolayers of Raw 264.7 cells (red). Infected Raw 264.7 cells were washed; fixed and extracellular spores were stained with Streptavidin-Alexa Fluor 350 conjugate (blue), stained for F-actin and analyzed by fluorescence microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043635#s2" target="_blank">Methods</a> section. Representative micrographs of phagocytosis of <i>C. difficile</i> spores are shown: A) Extracellular <i>C. difficile</i> spores (blue); B) Total <i>C. difficile</i> spores (green); C) Merged images. Bars represent 5 µm. White arrows highlight internalized spores.</p

Raw 264.7 cells bind and phagocytose <i>C. difficile</i> spores.

<p>A,B,C,D,E,F) SEM of Raw 264.7 cells infected with <i>C. difficile</i> spores under aerobic conditions. Note the active phagocytosis of the spores by Raw 264.7 cells in both panels. White arrows denote coiling phagocytosis. F) Transmission electron microscopy (TEM) of Raw 264.7 cells infected with <i>C. difficile</i> 630 spores at an MOI of 10 for 30 min under aerobic conditions. The area of adherence of <i>C. difficile</i> spores occurred at patchy regions at the end of protrusions from the surface of Raw 264.7 cells. Bar: 1 µm.</p

<i>C. difficile</i> spores remain intact inside the phagosome of Raw 264.7 cells.

<p>TEM images of Raw 264.7 cells infected with <i>C. difficile</i> spores under aerobic conditions (A–C, E, F) and with <i>B. subtilis</i> spores (D). TEM micrographs were taken after 30 min (A,B and D–F) and 24 h of infection. A) TEM shows that <i>C. difficile</i> spores are efficiently phagosytosed by Raw 264.7 cells. B), Phagosome containing <i>C. difficile</i> spores fuses with lysosomes, white arrows denotes fusion of lysosomes with the phagosome. C), <i>C. difficile</i> spores remain intact after 24 h of infection with Raw 264.7 macrophages. D) TEM micrograph of phagosytosed <i>B. subtilis</i> spores by Raw 264.7 cells. Phagosome’s membrane remains intact. E) TEM image showing phagocytosed <i>C. difficile</i> spore in a phagosome with membrane damage, white arrows denote disrupted phagosome membrane. F) TEM image rendering direct interactions between the surface of <i>C. difficile</i> spores and the phagosome’s membrane. White scale bar is 500 nm for panels A–C, and 100 m, for panels D-F.</p

Adherence and internalization of <i>C. difficile</i> spores by Raw 264.7 cells.

<p>Monolayers of Raw 264.7 cells were infected at an MOI of 10 with Alexa- and biotoin-labeled <i>C. difficile</i> spores of strains 630 and Pitt177, unbound spores washed, and samples prepared for fluorescence microscopy. Percentage of raw macrophages with at least one spore (A), number of spores per macrophage complex (B), and percentage of intracellular spores (C) were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043635#s2" target="_blank">Methods</a> section. D, E, F) The effect of cytochalasin D on the relative binding of spores to Raw 264.7 cells (D), relative number of spores per Raw-spore complex (E), and relative percentage of internalization (F) was evaluated without (white bars) and with 1 µM cytochalasin D (grey bars). Relative values refer to the relative percentage of Raw 264.7 cells with at least one spore (D), relative number of spores per Raw-spore complex (E), and relative percentage of internalization (F) in presence of cytochalasin D normalized to the culture medium control. Results are combined from at least three independent experiments and error bars are standard error of the mean. Asterisks (*) denote statistical difference at p<0.05, and double asterisks (**) denote statistical difference at p<0.01 compared to culture medium control.</p

Effect of microalgae on intestinal inflammation triggered by soybean meal and bacterial infection in zebrafish

<div><p>Soybean meal has been used in many commercial diets for farm fish; despite this component inducing intestinal inflammation. On the other hand, microalgae have increasingly been used as dietary supplements in fish feed. Nevertheless, the vast quantity of microalgae species means that many remain under- or unstudied, thus limiting wide scale commercial application. In this work, we evaluated the effects to zebrafish (<i>Danio rerio</i>) of including <i>Tetraselmis sp</i> (Ts); <i>Phaeodactylum tricornutum</i> (Pt); <i>Chlorella sp</i> (Ch); <i>Nannochloropsis oculata</i> (No); or <i>Nannochloropsis gaditana</i> (Ng) as additives in a soybean meal-based diet on intestinal inflammation and survival after <i>Edwardsiella tarda</i> infection. In larvae fed a soybean meal diet supplemented with Ts, Pt, Ch, or Ng, the quantity of neutrophils present in the intestine drastically decreased as compared to larvae fed only the soybean meal diet. Likewise, Ts or Ch supplements in soybean meal or fishmeal increased zebrafish survival by more than 20% after being challenged. In the case of Ts, the observed effect correlated with an increased number of neutrophils present at the infection site. These results suggest that the inclusion of Ts or Ch in fish diets could allow the use of SBM and at the same time improve performance against pathogen.</p></div

Microalgae improve neutrophil behavior after infection.

<p>(A) Assay strategy. (B) Accumulated mortality in challenged (continuous line) and unchallenged (doted line) Tg(BACmpo:GFP)ⁱ¹¹⁴ larvae fed with 100FM or 50SBM with and without <i>Tetraselmis</i> sp. (Ts). Mortality was monitored every 12 h until 60 hours post challenge (hpc). (C) Amount of intestinal neutrophils before challenge (T0), and at 60 hpc (T60 <i>Challenge</i>). At least 25 larvae per condition were analyzed in each three independent experiments. Statistical analysis was conducted using a non-parametric Mann-Whitney test. *<i>P</i> < 0.5, ***<i>P</i> < 0.001. Red bars represent the mean, and gray bars represent standard deviation.</p

Ingredients and nutrient composition of control and experimental diets.

<p>Ingredients and nutrient composition of control and experimental diets.</p

Effect of microalgae on intestinal inflammation.

<p>(A-G) Lateral view of 9 dpf Tg(BACmpo:GFP)ⁱ¹¹⁴ larvae after four days of feeding with the different diets; fishmeal (100FM), soybean meal (50SBM), and soybean meal + microalgae: 50SBM+Ch, 50SBM+Ts, 50SBM+A3Ng, 50SBM+Pt, or 50SBM+No. Black arrowheads indicate neutrophils. (H) The amount of intestinal neutrophils was quantified by immunohistochemistry against GFP. At least 25 larvae per diet were analyzed in each three different experiments. Statistical analysis was conducted using a non-parametric one-way ANOVA. ***<i>P</i> < 0.0001. Red bars represent the mean, and gray bars represent standard deviation.</p

Effect of microalgae on fish response to <i>Edwarsiella tarda</i> infection.

<p>Tg(BACmpo:GFP)ⁱ¹¹⁴ larvae fed with experimental or control diets were challenged with <i>E</i>. <i>tarda</i> for 5 h. Mortality was monitored every 12 h until 96 hour post challenge (hpc). Unchallenged larvae were subjected to the same feeding strategy and monitored during the same period. The graph represents the result obtained from three independent experiments. Statistical analysis was performed using survival curve analysis with Log-rank test against the 100FM and 50SBM diets. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. Solid lines represent challenged larvae and dotted lines represent control (unchallenged) larvae.</p