Cranberry extract inhibits in vitro adhesion of F4 and F18+Escherichia coli to pig intestinal epithelium and reduces in vivo excretion of pigs orally challenged with F18+ verotoxigenic E. coli.

F4+E. coli and F18+E. coli infections are an important threat for pig industry worldwide. Antibiotics are commonly used to treat infected piglets, but the emerging development of resistance against antibiotics raises major concerns. Hence, alternative therapies to prevent pigs from F4+E. coli and F18+E. coli infections need to be developed. Since cranberry previously showed anti-adhesive activity against uropathogenic E. coli, we aimed to investigate whether cranberry extract could also inhibit binding of F4+E. coli and F18+E. coli to pig intestinal epithelium. Using the in vitro villus adhesion assay, we found that low concentrations of cranberry extract (20µg or 100µg/ml) have strong inhibitory activity on F4+E. coli (75.3%, S.D.=9.31 or 95.8%, S.D.=2.56, respectively) and F18+E. coli adherence (100% inhibition). This effect was not due to antimicrobial activity. Moreover, cranberry extract (10mg or 100mg) could also abolish in vivo binding of F4 and F18 fimbriae to the pig intestinal epithelium in ligated loop experiments. Finally, two challenge experiments with F18+E. coli were performed to address the efficacy of in-feed or water supplemented cranberry extract. No effect could be observed in piglets that received cranberry extract only in feed (1g/kg or 10g/kg). However, supplementation of feed (10g/kg) and drinking water (1g/L) significantly decreased excretion and diarrhea. The decreased infection resulted in a decreased serum antibody response indicating reduced exposure to F18+E. coli.

Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

Optimization of a small intestinal segment perfusion model for heat-stable enterotoxin A induced secretion in pigs.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of infectious diarrhea both in human and pigs. After ingestion of contaminated food or water, ETEC bacteria colonize the small intestine where they produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, which induce watery diarrhea. We investigated the possibility of eliciting STa-induced secretion in jejunal segments of anesthetized pigs using a small-intestinal segment perfusion (SISP) model. Five consecutive mid-jejunal segments of anaesthetized piglets were perfused for 6h with different concentrations of STa in a physiologic salt solution. Changes in intestinal net fluid absorption were measured. From the results we could conclude that the STa response was dose-dependent and that continuous perfusion with 50 nM of STa or more was required to reduce net absorption. This concentration was sufficient to reduce net absorption compared to control segments in 12 out of 14 piglets. STa-induced responses however showed relative high variation between different jejunal segments of one pig, similar to the inter-segment variation seen in control animals where segments were perfused with physiologic salt solution. These results indicate that more optimization is required before this model could be used to test compounds that could interfere with the STa-induced fluid secretion.

Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

Immunomodulation by genetically engineered lactic acid bacteria.

The taxonomically diverse lactic acid bacteria (LAB) are unified by their capability to produce lactic acid from carbohydrates by fermentation. The LAB Lactococcus (L.) lactis has been characterized into great detail and is increasingly used as a production host for heterologous proteins. L. lactis is a non-pathogenic and non-colonizing LAB species and can be efficiently engineered to produce proteins of viral, bacterial or eukaryotic origin, both intra- or extracellularly. Importantly, orally formulated L. lactis strains (ActoBiotics), engineered to synthesize and secrete therapeutic peptides and proteins in the gastrointestinal tract, are already in advanced stages of preclinical and clinical development. This review focuses on the genetic engineering of LAB in general and L. lactis in specific to secrete high-quality, correctly processed, bioactive molecules derived from a eukaryotic background. The therapeutic applications of these genetically modified strains are discussed, as well as the need for a sound environmental containment strategy, and a detailed review is presented on Lactococcus strains engineered to produce specific antigens, antibodies, cytokines and trefoil factors, with special regards to immunomodulation.