Clostridium perfringens epsilon toxin increases the small intestinal permeability in mice and rats

Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel. © 2009 Goldstein et al

Transmission electron microscopy of control and epsilon toxin treated small intestinal loops displaying predominantly apoptotic-like changes.

<p>In control loops (A), enterocytes with normal looking nucleus and organelles are seen (scale bar = 15 µm). In epsilon treated loops (B) fibroblast with fragmented nucleus and edema with a polymorfonuclear cell (eosinophils) is seen; note as well, the electron lucent gaps between the epithelial cells (scale bar = 10 µm). (C) Abnormal looking red-blood cells together with degenerating cells, some displaying organelle and nuclear fragmentation (scale bar = 4 µm, or (D) cytoplasmatic (see in the inlet an apoptotic cell surrounded by another cells in process of nuclear fragmentation, probably at an early apoptosis stage) and (E) nuclear condensation (scale bar = 10 and 4 µm). (F) Lymphocyte in contact with a mast-cell (upper-left corner) together with some degenerating fibroblasts (scale bar = 4 µm).</p

Effect of ETX injected intravenously in mice in the enteropooling values.

<p>Six mice per treatment were injected intravenously with ETX diluted in peptone water. Mice were monitored during 1 hour and euthanized at the end of the experiments. Enteropooling was assessed after remotion of the small intestine and determination of wet and dried weight. Final values were relativized to the intestinal length and expressed as mg/cm.</p

<i>In vitro</i> characterization of the effects of <i>C. perfringens</i> epsilon toxin in the electrical parameters of the murine small intestine.

<p>Epsilon toxin was incubated in the mucosal side of ileal sheets mounted in modified Ussing chambers. (A) Short circuit current (<i>I_sc</i>) and (B) resistance (<i>Rt</i>) parameters were recorded each 10 minutes in tissues from 5 mice. (C) <i>Rt</i> values of ileal sheets incubated in the serosal side with 8,000 LD₅₀/ml of epsilon toxin. Each bar represents results for 4 mice. Results are expressed as means±SEM.</p

Mucosal ETX binding.

<p>Immunofluorescent detection of ETX (A) was negative in control tissues treated only with the vehicle solution. (B) ETX (2,000 LD₅₀/ml) treated small intestinal segments gave a clear signal in the tips of the villi and (C) lower in the crypts.</p

<i>Ex vivo</i> characterization of the effects of <i>C. perfringens</i> epsilon toxin in the electrical parameters of the murine small intestine.

<p>Intestinal loops were performed in groups of 4–6 anesthetized mice and injected with different concentrations of epsilon toxin in Ringer solution. In a particular set of experiments, intestines were removed after 3 hours of toxin injection and samples were mounted in Ussing chamber to measure changes in (A) short circuit current (<i>I_sc</i>) and (B) resistance (<i>Rt</i>). Those tissues were exposed to (C) luminal glucose or (D) serosal teophylline and the <i>I_sc</i> changes were recorded. In another set of experiments, ileal loops were injected with 1,000 LD₅₀ of epsilon toxin and incubated during different periods of time. Values of (E) <i>I_sc</i> or (F) <i>Rt</i> were recorded for those intestinal samples. Data are expressed as mean±SEM.</p

Enteropooling of mice inoculated intragastrically with different ETX concentrations.

<p>Six mice per treatment were inoculated intragastrically with ETX diluted in 1.5% PBS NaHCO₃ solution. Mice were monitored during 3 hours and euthanized at the end of the experiments. Enteropooling was assessed after removal of the small intestine and determination of wet and dried weight. Final values were relativized to the intestinal length and expressed as mg/cm.</p

<i>C. perfringens</i> epsilon toxin alters fluid homeostasis in the small intestine.

<p>(A) The enteropooling assay detected small intestinal fluid accumulation induced by luminal enterotoxin. Groups of 4 mice were orally administered with different doses of <i>C. botulinum</i> C2 toxin and intestinal fluid was determined 6 hours after oral administration. The results shown are the mean±standard error of the mean. (B) Epsilon toxin of <i>C. perfringens</i> altered fluid homeostasis in the mouse small intestine. Enteropooling was measured in groups of 4 mice treated with toxin (1,000 LD₅₀) or vehicle solution 2 and 6 h after oral administration. The results are expressed as the mean±standard error of the mean. (C) Epsilon toxin of <i>C. perfringens</i> produced fluid accumulation in mouse intestinal loops. Vehicle solution with or without 1,000 LD₅₀ of toxin were injected in ligated ileal segments. The loops were excised 3 hours after injection of the toxin and intestinal water was determined gravimetrically. Each point represents the mean±standard error. (D) Basal absorption of water by the mouse small intestine was diminished by <i>C. perfringens</i> epsilon toxin when measured by the single-pass perfusion method. Perfusion began with an equilibration period of 45 min. Then, Ringer solution containing 500 LD₅₀/ml of epsilon toxin or Ringer solution alone was perfused and after 60 minutes four 15-min samples were collected. The volume of each sample was determined. Each mouse was killed at the end of the perfusion, and the segment of jejunum was removed and its length measured. The absorbed or secreted volume was calculated as collected volume minus perfused volume and expressed per centimeter of perfused bowel per minute. Data were relativized to the value obtained at time 15. Changes become statistically significant after 45 minutes of toxin delivery (<i>P</i><0.05).</p