Michaela Loos

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.

Mucosal priming of the murine immune system against enterohemorrhagic Escherichia coli O157:H7 using Lactococcus lactis expressing the type III secretion system protein EspB

Enterohemorrhagic Escherichia coli (EHEC), particularly E. coli serotype O157:H7, has been responsible for multiple human outbreaks of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Humans become infected by direct or indirect contact with faeces of asymptomatic EHEC shedding ruminants. Currently there is no human or animal vaccine available against EHEC infection. EHEC use a type III secretion system (T3SS) to colonize the intestine and therefore eliciting mucosal immunity against T3SS proteins could be a potential vaccination strategy. To develop such a mucosal vaccine, EspB - a significant member of the T3SS - was intracellularly expressed in Lactococcus lactis (LL-EspB) and this strain was used to immunize BALB/c mice orally. Ten days post-immunization, no specific antibody response was detected in serum or faeces of immunized mice. However, statistically significant (P<0.0001) levels of specific serum Ig and faecal IgA were detected after intraperitoneal boosting of the orally immunized mice with recombinant EspB. Our results show that oral administration of LL-EspB resulted in mucosal priming of BALB/c mice against the EHEC T3SS protein, EspB. Nevertheless, an optimized EspB expression in L. lactis may be required to improve the mucosal immune response.

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.

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.