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.

We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

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Enterohemorrhagic Escherichia coli (EHEC) have been responsible for several outbreaks of hemolytic-uremic syndrome (HUS) worldwide. HUS is the most common cause of acute renal failure in children and results in fatalities as high as 50% in the elderly. Currently, neither a specific treatment nor a vaccine is available for EHEC. Lactococcus lactis is a generally regarded as safe "GRAS" bacterium that offers a valuable platform for oral vaccine delivery. Toward the development of an oral vaccine against EHEC, we have previously constructed a recombinant L. lactis strain expressing the EHEC antigen, EspB in the cytoplasmic compartment. However, oral immunization of mice with this strain induced weak priming of the immune system. This outcome was attributed to the rather low levels of EspB expressed by this recombinant strain. Therefore, in the present study we optimized the expression of EspB in L. lactis by secreting the antigen either under constitutive or nisin-inducible control. Indeed, oral immunization of mice with the EspB-secreting strains successfully induced specific mucosal and systemic antibody responses. These responses were associated with mixed Th1/Th2 cell responses in Peyer's Patches and mesenteric lymph nodes. Moreover, immunized mice exhibited significant protection against E. coli O157:H7 colonization, as indicated by the reduced amount and/or duration of the bacterial fecal shedding. Our results demonstrate the protective potential of EspB as an oral vaccine against EHEC infection. Additionally, the study demonstrates the efficient delivery of recombinant EspB by the "GRAS" bacterium, L. lactis. The safety profile of L. lactis as a vaccine vehicle can particularly be beneficial to children and elderly as high-risk groups for HUS incidence.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of dehydrating diarrhoea in children and weaned piglets living under subhygienic conditions. After colonization of the small intestine, ETEC produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Individual human ETEC strains generally produce only one type of enterotoxin, and in symptomatic patients from developing countries the heat-stable enterotoxin A (STa) producing strains seem by far the most prevalent. STa binds to and activates guanylyl cyclase C (GC-C) in the brush border of intestinal epithelium. An increase in cGMP in turn activates cGMP-dependent protein kinase II (cGKII) leading to phosphorylation of the cystic fibrosis transmembrane regulator (CFTR) driving Cl-secretion and inhibition of NaCl absorption followed by net loss of water through osmotic diarrhoea. We used a small-intestinal segment perfusion model of ETEC diarrhea in pigs [1] in which we investigated the possibility of eliciting STa induced secretion. Five consecutive mid-jejunal segments of anaesthetized piglets were perfused for 6 hours with different concentrations of STa (150-0.62 nM) in a physiologic salt solution. Changes in intestinal net fluid absorption were measured. We concluded that the STa response was dose-dependent and that continuous perfusion with 50 nM of STa or more was required to reduce net absorption. In all piglets the control segments showed net absorption. Fifty nanomolar of STa was sufficient to reduce net absorption compared to control segments in 10 out of 12 piglets; in 8 of them net absorption was reversed to net secretion. STa-induced responses showed relative high variation between different jejunal segments similar to the inter-segment variation seen in control animals where segments were perfused with physiologic salt solution. 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. 1. Nabuurs, M.J., et al., A long-term perfusion test to measure net absorption in the small intestine of weaned pigs. Res Vet Sci, 1993. 55(1): p. 108-14.